Submicroscopic chromosomal imbalances in idiopathic Silver-Russell syndrome (SRS): the SRS phenotype overlaps with the 12q14 microdeletion syndrome.

Silver-Russell syndrome (SRS) is a heterogeneous disorder associated with intrauterine and postnatal growth restriction, body asymmetry, a relative macrocephaly, a characteristic triangular face and further dysmorphisms. In about 50% of patients, genetic/epigenetic alterations can be detected: >38% of patients show a hypomethylation of the IGF2/H19 imprinting region in 11p15, whereas the additional 10% carry a maternal uniparental disomy of chromosome 7. In single cases, cytogenetic aberrations can be detected. Nevertheless, there still remain 50% of SRS patients without known genetic/epigenetic alterations. To find out whether submicroscopic imbalances contribute to the aetiology of SRS, 20 idiopathic SRS patients were screened with the Affymetrix GeneChip Human Mapping 500 K array set. Apart from known apathogenic copy number variations, we identified one patient with a 12q14 microdeletion. The 12q14 microdeletion syndrome is characterised by dwarfism but it additionally includes mental retardation and osteopoikilosis. The deletion in our patient is smaller than those in the 12q14 microdeletion carriers but it also affects the LEMD3 and the HMGA2 genes. LEMD3 haploinsufficiency and point mutations have been previously associated with osteopoikilosis but radiographs of our patient at the age of 16 years did not reveal any hint for osteopoikilosis lesions. Haploinsufficiency of HMGA2 is probably responsible for aberrant growth in 12q14 microdeletion syndrome. However, in this study, a general role of HMGA2 mutations for SRS was excluded by sequencing of 20 idiopathic patients. In conclusion, our results exclude a common cryptic chromosomal imbalance in idiopathic SRS patients but show that chromosomal aberrations are relevant in this disease. Thus, molecular karyotyping is indicated in SRS and should be included in the diagnostic algorithm.

Use of multiplex ligation-dependent probe amplification increases the detection rate for 11p15 epigenetic alterations in Silver-Russell syndrome.

Silver-Russell syndrome (SRS) describes a malformation syndrome with severe intrauterine and postnatal growth retardation. Currently, two major (epi)mutations have been described: while approximately 10% of patients carry a maternal uniparental disomy of chromosome 7 (UPD7), 35-60% show a hypomethylation at the H19 differentially methylated regions (DMRs) in 11p15. Until recently, a Southern-blot based test was routinely used to identify epimutation carriers. Nevertheless, this test was time consuming and hampered by the huge amount of genomic DNA needed. With the methylation-specific multiplex ligation-dependent probe amplification assay (MLPA) for SRS, a PCR-based test is now available, allowing the analysis also of small amounts of DNA. Probes in this assay hybridize to the H19 DMRs but do not cover the genomic target of the Southern-blot probe. We now screened 72 patients with SRS by MLPA. Hypomethylation of the H19 DMRs was confirmed in all patients analyzed by Southern blot. In addition, we identified six individuals with hypomethylation of the H19 DMR who had previously normal blot results. This discrepancy can be explained by the observed generally lower degree of demethylation in this group, possibly not detectable by the less sensitive Southern-blot method but also with a varying degree of methylation at different DMRs in the same individual. Apart from hypomethylation in the H19 DMR, we observed a slight demethylation for one of the IGF2 probes. The total detection rate of 11p15 hypomethylation is now increased to >38%. Considering maternal UPD7 and chromosomal aberrations, (epi)genetic alterations now account for more than 50% of SRS patients. In summary, MLPA represents an easy, low cost and reliable system in the molecular diagnostics of SRS.

ICR1 epimutations in llp15 are restricted to patients with Silver-Russell syndrome features.

(Epi)mutations affecting chromosome llp15 are well known to be associated with growth disturbances. The finding of llp15 mutations in the overgrowth disease Beckwith-Wiedemann syndrome led to the identification of imprinted growth-promoting genes which are expressed paternally and of imprinted growth-suppressing genes in the same region that are expressed maternally. An opposite epimutation in the same region is associated with Silver-Russell syndrome (SRS), a growth retardation disorder characterised by a typical facial gestalt, clinodactyly V and asymmetry. In more than 30% of patients with SRS, hypomethylation of the telomeric llp15 imprinting domain (ICR1) can be detected. However, the general significance of this epimutation for human growth retardation was unclear. In a previous study' we showed that the ICR1 epimutation is not present in growth retarded patients with dysmorphisms not typical for SRS, but its role in the development of isolated growth restriction needed to be further elucidated. We therefore screened 30 patients with isolated pre- and postnatal growth retardation (IUGR/PNGR) and 65 patients diagnosed with SRS by external clinicians for ICR1I epimutations. In the latter group clinical data were rarely provided. These 65 'SRS' patients were additionally analysed for maternal uniparental disomy 7 (matUPD7). We excluded ICR1 hypomethylation in all 30 patients with isolated growth retardation. In the SRS group, we detected four cases with ICR1 epimutation and three with matUPD7. By combining our data with those from our previous study we could show that the hypomethylation of ICR1 in llp15 is indeed restricted to patients with SRS features and can be disregarded in isolated IUGR/PNGR. Thus, testing for the epimutation is indicated only in case of growth restriction in association with clinical signs reminiscent of SRS. The low detection rate of the ICR1 epimutation in our 'SRS' group can be explained by the clinical heterogeneity of cases referred by external institutions.

Epigenetic mutations in 11p15 in Silver-Russell syndrome are restricted to the telomeric imprinting domain.

INTRODUCTION: Silver-Russell syndrome (SRS; also know as Russell-Silver syndrome) is a heterogeneous syndrome which is characterised by severe intrauterine and postnatal growth retardation and typical dysmorphic features. Recently, the first SRS patients with (epi)genetic mutations in 11p15 affecting the telomeric imprinting domain have been identified. Interestingly, opposite mutations are associated with Beckwith-Wiedemann syndrome (BWS). However, the general significance of epigenetic mutations in 11p15 for the aetiology of SRS remained unclear. METHODS: We screened a cohort of 51 SRS patients for epimutations in ICR1 and KCNQ1OT1 by methylation sensitive Southern blot analyses. RESULTS: ICR1 demethylation could be observed in 16 of the 51 SRS patients, corresponding to a frequency of approximately 31%. Changes in methylation at the KCNQ1OT1 locus were not detected. DISCUSSION: Combining these data with those on maternal duplications in 11p15, nearly 35% of SRS cases are associated with detectable (epi)genetic disturbances in 11p15. We now have to also consider a general involvement of 11p15 alterations in growth retarded patients with only minor or without further dysmorphic features. SRS and BWS may now be regarded as two diseases caused by opposite (epi)genetic disturbances of the same chromosomal region displaying opposite clinical pictures.

(Epi)mutations in 11p15 significantly contribute to Silver-Russell syndrome: but are they generally involved in growth retardation?

(Epi)mutations affecting chromosome 11p15 are meanwhile well known to be associated with growth disturbances. The finding of 11p15 mutations in the overgrowth disease Beckwith-Wiedemann syndrome (BWS) led to the identification of imprinted growth-promoting genes which are expressed paternally and of imprinted growth-suppressing genes in the same region that are expressed maternally. Recently, the opposite (epi)mutations of the same region have been reported to result in growth retardation: maternal duplications of 11p15 as well as hypomethylation of the telomeric 11p15 imprinting domain (ICR1) could be identified in patients with Silver-Russell syndrome (SRS), a disease which is in particular characterised by intrauterine and postnatal growth retardation. To elucidate whether 11p15 mutations are generally involved in growth retardation we screened 125 growth retarded patients, among them 47 patients with SRS-like features and 20 with isolated growth retardation. Additional 58 patients were presented with clinical signs not consistent with SRS. We excluded 11p15 duplications in all 123 families by short tandem repeat typing. ICR1 hypomethylation was investigated by Southern-blot analyses and was therefore restricted to samples with a large amount of DNA. We identified ICR1 hypomethylation in 20% of the patients with SRS-like features (n=25). No further cases were detectable in the other two subgroups with isolated growth retardation (n=20) and with clinical signs not consistent with SRS (n=23), respectively. Our data show that 11p15 duplications are rare in growth retardation in general and that they seem to be restricted to patients with SRS features. Furthermore, testing for the ICR1 hypomethylation should also be focused on patients with SRS features. While the ICR1 epimutation is detectable with a significant frequency only in SRS patients, its role for isolated growth retardation remains to be elucidated.