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Decreased expression of cell proliferation-related genes in clonally derived skin fibroblasts from children with Silver-Russell syndrome is independent of the degree of 11p15 ICR1 hypomethylation.

Heckmann D, Urban C, Weber K, Kannenberg K, Binder G - Clin Epigenetics (2015)

Bottom Line: Cell proliferation was diminished in SRShypo compared to Cnormo (p = 0.035).The analysis of severely ICR1 hypomethylated clonal fibroblasts did not reveal functional differences compared to normomethylated clones with respect to IGF2 and H19 expression.A difference compared to the clones from healthy individuals was present in the form of a lower proliferation rate, presumably due to impaired cell cycle progression.

View Article: PubMed Central - PubMed

Affiliation: Pediatric Endocrinology, University Children's Hospital Tuebingen, Hoppe-Seyler-Straße 1, 72076 Tuebingen, Germany.

ABSTRACT

Background: The in vitro analysis of the hypomethylation of imprinting control region 1 (ICR1) within the IGF2/H19 locus is challenged by the mosaic distribution of the epimutation in tissues from children with Silver-Russell syndrome (SRS). To exclude mosaicism, clonal cultures of skin fibroblasts from four children with SRS and three controls were analyzed. Cell proliferation, IGF-II secretion, and IGF2 and H19 expression were measured, and a microarray expression analysis was performed.

Results: Single-cell expansion established severely ICR1 hypomethylated clones (SRShypo) and normomethylated clones (SRSnormo) from the patients and controls (Cnormo). IGF2 expression was below the detection limit of the quantitative real-time PCR (qRT-PCR) assay, whereas H19 expression was detectable, without differences between fibroblast clones. Cell count-related IGF-II release was comparable in SRShypo and Cnormo supernatants. Cell proliferation was diminished in SRShypo compared to Cnormo (p = 0.035). The microarray analysis revealed gene expression changes in SRS clones, predicting a decrease in cell proliferation and a delay in mitosis.

Conclusions: The analysis of severely ICR1 hypomethylated clonal fibroblasts did not reveal functional differences compared to normomethylated clones with respect to IGF2 and H19 expression. A difference compared to the clones from healthy individuals was present in the form of a lower proliferation rate, presumably due to impaired cell cycle progression.

No MeSH data available.


Related in: MedlinePlus

Differential analysis results. (a) Venn diagram of differentially regulated genes in the SRShypo, SRSnormo, and Cnormo clones. (b) Differentially regulated genes in the SRShypo, SRSnormo, and Cnormo groups. Clustering heatmap based on multigroup comparison using the statistical parameters σ = 0.174, p = 0.05, and q = 0.529103 in Qlucore Omics Explorer 3.0. Ninety-two genes were significantly differentially expressed between the three groups. The analysis of the SRSnormo clone S1lo_7 is not shown. (c) Differentially expressed PRUNE2 in the SRShypo, SRSnormo, and Cnormo groups, as validated by qRT-PCR. qRT-PCR results were normalized to HepG2 cell gene expression. PRUNE2 is expressed at a significantly lower level in SRShypo compared to Cnormo (p = 0.017). (d) Differentially expressed IL6 in the SRShypo, SRSnormo, and Cnormo groups, as validated by qRT-PCR. qRT-PCR results were normalized to HepG2 cell gene expression. IL6 is expressed at a significantly higher level in SRShypo compared to Cnormo (p = 0.009).
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Fig4: Differential analysis results. (a) Venn diagram of differentially regulated genes in the SRShypo, SRSnormo, and Cnormo clones. (b) Differentially regulated genes in the SRShypo, SRSnormo, and Cnormo groups. Clustering heatmap based on multigroup comparison using the statistical parameters σ = 0.174, p = 0.05, and q = 0.529103 in Qlucore Omics Explorer 3.0. Ninety-two genes were significantly differentially expressed between the three groups. The analysis of the SRSnormo clone S1lo_7 is not shown. (c) Differentially expressed PRUNE2 in the SRShypo, SRSnormo, and Cnormo groups, as validated by qRT-PCR. qRT-PCR results were normalized to HepG2 cell gene expression. PRUNE2 is expressed at a significantly lower level in SRShypo compared to Cnormo (p = 0.017). (d) Differentially expressed IL6 in the SRShypo, SRSnormo, and Cnormo groups, as validated by qRT-PCR. qRT-PCR results were normalized to HepG2 cell gene expression. IL6 is expressed at a significantly higher level in SRShypo compared to Cnormo (p = 0.009).

Mentions: Microarray gene expression analyses were performed to profile differentially expressed genes. In total, 587 genes were found to be differently expressed in SRShypo compared to Cnormo, with 522 genes in SRSnormo compared to Cnormo (Figure 4a). The number of differentially expressed genes was significantly lower when comparing SRShypo and SRSnormo (n = 117). In the array, the expression of IGF2 and H19 was not significantly deregulated between groups of SRShypo, SRSnormo, and Cnormo.Figure 4


Decreased expression of cell proliferation-related genes in clonally derived skin fibroblasts from children with Silver-Russell syndrome is independent of the degree of 11p15 ICR1 hypomethylation.

Heckmann D, Urban C, Weber K, Kannenberg K, Binder G - Clin Epigenetics (2015)

Differential analysis results. (a) Venn diagram of differentially regulated genes in the SRShypo, SRSnormo, and Cnormo clones. (b) Differentially regulated genes in the SRShypo, SRSnormo, and Cnormo groups. Clustering heatmap based on multigroup comparison using the statistical parameters σ = 0.174, p = 0.05, and q = 0.529103 in Qlucore Omics Explorer 3.0. Ninety-two genes were significantly differentially expressed between the three groups. The analysis of the SRSnormo clone S1lo_7 is not shown. (c) Differentially expressed PRUNE2 in the SRShypo, SRSnormo, and Cnormo groups, as validated by qRT-PCR. qRT-PCR results were normalized to HepG2 cell gene expression. PRUNE2 is expressed at a significantly lower level in SRShypo compared to Cnormo (p = 0.017). (d) Differentially expressed IL6 in the SRShypo, SRSnormo, and Cnormo groups, as validated by qRT-PCR. qRT-PCR results were normalized to HepG2 cell gene expression. IL6 is expressed at a significantly higher level in SRShypo compared to Cnormo (p = 0.009).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig4: Differential analysis results. (a) Venn diagram of differentially regulated genes in the SRShypo, SRSnormo, and Cnormo clones. (b) Differentially regulated genes in the SRShypo, SRSnormo, and Cnormo groups. Clustering heatmap based on multigroup comparison using the statistical parameters σ = 0.174, p = 0.05, and q = 0.529103 in Qlucore Omics Explorer 3.0. Ninety-two genes were significantly differentially expressed between the three groups. The analysis of the SRSnormo clone S1lo_7 is not shown. (c) Differentially expressed PRUNE2 in the SRShypo, SRSnormo, and Cnormo groups, as validated by qRT-PCR. qRT-PCR results were normalized to HepG2 cell gene expression. PRUNE2 is expressed at a significantly lower level in SRShypo compared to Cnormo (p = 0.017). (d) Differentially expressed IL6 in the SRShypo, SRSnormo, and Cnormo groups, as validated by qRT-PCR. qRT-PCR results were normalized to HepG2 cell gene expression. IL6 is expressed at a significantly higher level in SRShypo compared to Cnormo (p = 0.009).
Mentions: Microarray gene expression analyses were performed to profile differentially expressed genes. In total, 587 genes were found to be differently expressed in SRShypo compared to Cnormo, with 522 genes in SRSnormo compared to Cnormo (Figure 4a). The number of differentially expressed genes was significantly lower when comparing SRShypo and SRSnormo (n = 117). In the array, the expression of IGF2 and H19 was not significantly deregulated between groups of SRShypo, SRSnormo, and Cnormo.Figure 4

Bottom Line: Cell proliferation was diminished in SRShypo compared to Cnormo (p = 0.035).The analysis of severely ICR1 hypomethylated clonal fibroblasts did not reveal functional differences compared to normomethylated clones with respect to IGF2 and H19 expression.A difference compared to the clones from healthy individuals was present in the form of a lower proliferation rate, presumably due to impaired cell cycle progression.

View Article: PubMed Central - PubMed

Affiliation: Pediatric Endocrinology, University Children's Hospital Tuebingen, Hoppe-Seyler-Straße 1, 72076 Tuebingen, Germany.

ABSTRACT

Background: The in vitro analysis of the hypomethylation of imprinting control region 1 (ICR1) within the IGF2/H19 locus is challenged by the mosaic distribution of the epimutation in tissues from children with Silver-Russell syndrome (SRS). To exclude mosaicism, clonal cultures of skin fibroblasts from four children with SRS and three controls were analyzed. Cell proliferation, IGF-II secretion, and IGF2 and H19 expression were measured, and a microarray expression analysis was performed.

Results: Single-cell expansion established severely ICR1 hypomethylated clones (SRShypo) and normomethylated clones (SRSnormo) from the patients and controls (Cnormo). IGF2 expression was below the detection limit of the quantitative real-time PCR (qRT-PCR) assay, whereas H19 expression was detectable, without differences between fibroblast clones. Cell count-related IGF-II release was comparable in SRShypo and Cnormo supernatants. Cell proliferation was diminished in SRShypo compared to Cnormo (p = 0.035). The microarray analysis revealed gene expression changes in SRS clones, predicting a decrease in cell proliferation and a delay in mitosis.

Conclusions: The analysis of severely ICR1 hypomethylated clonal fibroblasts did not reveal functional differences compared to normomethylated clones with respect to IGF2 and H19 expression. A difference compared to the clones from healthy individuals was present in the form of a lower proliferation rate, presumably due to impaired cell cycle progression.

No MeSH data available.


Related in: MedlinePlus