Silver-Russell syndrome (SRS) is a heterogenous syndrome which is mainly associated with severe intrauterine and postnatal growth retardation. Typical facial dysmorphisms and further characteristic symptoms can additionally be observed. So far the clinical diagnosis can not be confirmed in all patients with SRS. ~38-63% of SRS patients carry (epi)genetic mutations in 11p15 and in ~7-10% defects on chromosome 7 can be detected. One of the main tasks of this doctoral thesis was the analyses of (epi)genetic changes in the chromosomal region 11p15 and the verification of their functional relevance for the etiology of SRS. Therefore methylation-specific analyses were carried out in 72 well characterised patients with SRS, 188 patients referred for routine SRS-diagnostics and additional 108 patients with isolated growth retardation. Recently Gicquel et al. (2005) firstly detected a hypomethylation of the ICR1 (Imprinting Control Region 1) in 11p15 in patients with SRS. Due to these observations we carried out Multiplex Ligation-dependant Probe Amplification analyses to determine the frequency of this aberration. While 44.4% of the well characterised SRS patients showed hypomethylation of the ICR1, in patients from routine SRS diagnostics a frequency of only 15.3% could be detected. None of the patients with isolated growth retardation showed a hypomethylation of 11p15, thereby confirming the assumption that this epimutation is specific for SRS. Further analyses of the results from this methylation specific assay did not reveal any other so far unknown (epi)genetic disturbances in 11p15, such as methylation defects in the ICR2. Hypomethylation of further imprinted regions in the genome additionally to the ICR1 could also not be detected in the analysed SRS patients. It is well known that some patients with SRS show a maternal duplication in 11p15 always including both imprinting control regions, ICR1 and ICR2. In this study we could detect the first case with a maternal duplication restricted to the ICR2 in 11p15 in a patient with SRS, thus making a relevance of imprinted factors in the ICR2 for the etiology of SRS probable. Investigations on the functional consequence of this duplication were carried out by analysing the expression of the growth relevant and imprinted factor CDKN1C and a further gene (KCNQ1) localised in the duplicated region. In addition to this case we could detect another patient with SRS showing a maternal duplication in 11p15, including both ICRs. Moreover, functional analyses on three variations of the genomic sequence in the SRS-candidate gene H19 have been performed. We therefore carried out expression analyses in cultured HEK293- and Cos7-cells after transfection with constructs containing the different variants. An altered splicing of the H19 mRNA in comparison to wild-type construct could be found in two of the three patients (SR81K; SR93K), therefore indicating a relevance of H19 for the etiology of SRS. In case of the patient SR93K analyses of RNA from lymphocytes could confirm the former results. In addition to the analyses for chromosome 11p15 we performed investigations on disturbances of chromosome 7. Two of the analysed SRS patients showed a segmental uniparental disomy of chromosome 7q, which is a very rare aberration and to date only reported in two further cases of SRS. Therefore, our results clearly support the relevance of this scarce genetic finding. Isolated methylation defects in the imprinted region 7q32 - which might have a functional role in SRS - could not be observed in 54 SRS patients without hypomethylation in 11p15. To determine a possible functional link between the different imprinted regions associated with SRS on chromosome 11 and 7, we carried out analyses to detect potential interchromosomal interactions between these regions. Multicolour-FISH analyses showed a colocalisation of the chromosomal region 11p15 with 7p12-p11.2 (GRB10) and 7q32 (PEG1/MEST) in single placental interphase nuclei. These interactions were always restricted to one allele, which might lead to the assumption that, due to the imprinting of these regions, the association is essential only between the two chromosomes of one defined parental origin. Furthermore quantitative analyses were carried out to show a possible influence of the protein CTCF on the expression of the genes localised in the SRS-candidate regions 7p12-p11.2 and 7q32. CTCF is a regulator of expression and imprinting of various genes, among others for the genes localised in the ICR1. Analyses in placental primary cell cultures clearly showed an inhibition of the PEG1/MEST expression by CTCF. Therefore a common mechanism for the regulation of these two SRS-associated regions is conceivable and might explain their link in the etiology of SRS.
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