Abstract
BackgroundThe 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.ResultsSingle-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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-014-0038-0) contains supplementary material, which is available to authorized users.
Highlights
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)
In addition to analyzing cell proliferation rates, we studied the expression of IGF2, H19, and miRNAs originating from the IGF2/H19 locus in normo- and severely hypomethylated clones
The methylation status of ICR1 and ICR2 at the IGF2/H19 locus was found to be stable between passages 4 and 17
Summary
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). Silver-Russell syndrome (SRS, OMIM 180860) is a sporadic, clinically, and genetically heterogeneous disorder characterized by severe intrauterine and postnatal growth failure, a typical triangular face, asymmetric growth of the body, relative macrocephaly, and underweight [1]. The expression of the genes IGF2 and H19 is allelespecific due to imprinting. ICR1 on 11p15 controls the expression of the imprinted genes IGF2 and H19. It is thought that hypomethylation of the paternal ICR1 allele might result in the reduced production of the important fetal growth factor IGF-II [7]. Hypermethylation on the maternal allele in Beckwith-Wiedemann syndrome might result in increased IGF-II production and a predisposition toward tumor growth [8]. H19 is highly expressed from the early stages of embryogenesis to fetal life in many organs including the fetal adrenal, liver, and placenta tissues but is nearly completely downregulated postnatally [11]
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