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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

Proliferation rates of SRShypo clones and Cnormo clones. Fibroblast clones were cultured for 14 days (d) in standard medium. Proliferation of four different SRShypo clones (S1sh_12, S2lo_7, S2lo_1, S5sh_11) and three different Cnormo clones (K2li_17, K3li_3, K3re12) was measured using the MTT assay after 1, 7, and 14 days.
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Fig1: Proliferation rates of SRShypo clones and Cnormo clones. Fibroblast clones were cultured for 14 days (d) in standard medium. Proliferation of four different SRShypo clones (S1sh_12, S2lo_7, S2lo_1, S5sh_11) and three different Cnormo clones (K2li_17, K3li_3, K3re12) was measured using the MTT assay after 1, 7, and 14 days.

Mentions: Cell proliferation during the 14 days of culture, as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, was slightly lower in the SRShypo group (S1sh_12, S2lo_7, S2lo_1, S5sh_11) than in the Cnormo group (K2li_17, K3li_3, K3re_12) after 7 days and significantly lower after 14 days of culture (Figure 1) (p = 0.035). Cell proliferation slowed after 1 week of culture in the Cnormo clones and stagnated in the SRShypo clones.Figure 1


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)

Proliferation rates of SRShypo clones and Cnormo clones. Fibroblast clones were cultured for 14 days (d) in standard medium. Proliferation of four different SRShypo clones (S1sh_12, S2lo_7, S2lo_1, S5sh_11) and three different Cnormo clones (K2li_17, K3li_3, K3re12) was measured using the MTT assay after 1, 7, and 14 days.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4318184&req=5

Fig1: Proliferation rates of SRShypo clones and Cnormo clones. Fibroblast clones were cultured for 14 days (d) in standard medium. Proliferation of four different SRShypo clones (S1sh_12, S2lo_7, S2lo_1, S5sh_11) and three different Cnormo clones (K2li_17, K3li_3, K3re12) was measured using the MTT assay after 1, 7, and 14 days.
Mentions: Cell proliferation during the 14 days of culture, as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, was slightly lower in the SRShypo group (S1sh_12, S2lo_7, S2lo_1, S5sh_11) than in the Cnormo group (K2li_17, K3li_3, K3re_12) after 7 days and significantly lower after 14 days of culture (Figure 1) (p = 0.035). Cell proliferation slowed after 1 week of culture in the Cnormo clones and stagnated in the SRShypo clones.Figure 1

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