Implementierung hochauflösender molekulargenetischer Methoden zur Klärung der Pathophysiologie des Silver-Russell-Syndroms

Silver Russell syndrome (SRS; OMIM #180860) is a clinically and genetically heterogeneous imprinting disorder characterized by severe pre- and postnatal growth retardation, a relative macrocephaly, a triangular face, asymmetry of the body and/or the limbs, and a clinodactyly of the fifth digits. A molecular genetic confirmation of the clinical diagnosis is currently feasible in approximately half of the patients: While in 7-10% of patients a maternal uniparental disomy of chromosome 7 (upd(7)mat) is detectable, in ca. 40% of patients a hypomethylation of the ICR1 in 11p15 can be observed. The aim of this study was to contribute to the understanding of the pathophysiology of the SRS by implementation of high resolution molecular genetic techniques. For this purpose analyses of genomic mutations, methylation-specific tests as well as microarray analyses were performed in a study cohort of 42 patients with clinical diagnosis of SRS but unknown etiology (“idiopathic SRS”) and 31 SRS patients with ICR1-hypomethylation. Additionally, two single patients from SRS routine diagnostics carrying (epi)mutations on chromosome 7 were analysed. Due to reports in the literature indicating a pathogenic relevance for the SRS (Mackay et al., 2008; Shiura et al., 2009; Lynch et al., 2011) the genes ZFP57 and HMGA2 as well as the GRB10-CPGI2 were screened for genomic mutations. However, in the patients analysed in this study no pathogenic variants were detectable (Spengler et al., 2009; 2010). Thus a relevant causal relationship between mutations in these factors and SRS is unlikely. The identification of (epi)mutations at imprinted loci in approximately 50% of SRS patients leads to the assumption that at least some of the idiopathic patients also carry epimutations which are not detectable with the currently applied analysis methods. Therefore, on the one hand methylation analyses in a tissue not routinely tested so far were performed in this study. Furthermore, new methods for the detection of epimutations in the SRS patients were developed and established. For the identification of tissue-specific mosaicism buccal smear DNA of SRS patients with epimutation and idiopathic SRS patients was analysed in addition to the routinely tested lymphocyte DNA. The results in lymphocyte DNA of both patient subgroups could be confirmed in buccal smear DNA (Spengler et al., 2011). Therefore it can be assumed that the epimutation always affects both tissues. For the detection of upd(7)mat as well as isolated methylation defects a methylation-specific (MS)-PCR for the MEST locus in 7q32 was used in the past. In this study an additional MS-PCR for the identification of segmental uniparental disomies as well as epimutations at the GRB10 locus in 7p12 was established. An isolated hypermethylation was identified in one out of 31 patients; therefore it can be assumed that this type of epimutation is a rare finding in SRS. Due to the observation of multilocus methylation defects (MLMD) in patients with various imprinting disorders a MS-SNuPE (methylation-specific single nucleotide primer extension) assay was established for the detection of MLMD in SRS patients. In the future, this assay will allow the parallel analysis and quantification of methylation at the eight loci most commonly affected in MLMD in one approach. Furthermore the detection of low-level epimutation mosaicism, which escapes the currently used, less sensitive methods, is possible with this technique. To obtain evidence for new SRS candidate regions genome-wide analyses with DNA microarrays were performed. In first screening studies submicroscopic (< 3 Mb) imbalances had been identified in several patients with typical SRS phenotype (Bruce et al., 2010; Spengler et al., 2010). For the systematic detection of such imbalances in our own SRS cohort high resolution arrays as well as the necessary analysis software were implemented. Using the Affymetrix SNP6.0 array the 31 patients with ICR1-hypomethylation as well as the 42 idiopathic patients were analyzed. While in the group of the epimutation carriers no obviously clinically relevant aberration could be found, pathogenic submicroscopic imbalances were detected in a considerable proportion of idiopathic SRS patients (21.4%). In further 11.9% of patients copy number alterations with so far unknown clinical significance were identified. In summary, performing array analyses for the detection of submicroscopic imbalances is indicated in routine diagnostics after exclusion of the known (epi)mutations of chromosomes 7 and 11 in SRS- and SRS-like patients.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2011

Author dc:creator

• Spengler, Sabrina

Contributors dc:contributor

• Eggermann, Thomas

• info:eu-repo/classification/ddc/570
• Silver-Russell-Syndrom
• DNS-Chip
• Microarray
• Genetisches Imprinting
• Methylierung
• Molekulargenetik
• Chromosom 11
• Chromosomenanalyse
• Chromosomenaberration
• Epigenetik
• Biowissenschaften, Biologie
• MS-MLPA
• MS-SNuPE
• ICR1-Hypomethylierung
• Methylierungsanalysen
• upd(7)mat
• Silver-Russell syndrome
• genomic imprinting
• methylation analysis
• epigenetic

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-113974%22

OAI record oai:identifier

https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A51711