IGF2/H19 hypomethylation in Silver-Russell syndrome and isolated hemihypoplasia

Silver-Russell syndrome (SRS) is a clinically and genetically heterogeneous syndrome characterized by severe pre and postnatal growth retardation, body asymmetry and a typical facial phenotype with a triangular face and relative macrocephaly. In 30% of patients, the differentially methylated IGF2/H19 imprinting center region (ICR1) on chromosome 11p15 was found to be hypomethylated, as determined by Southern blot analysis of an HpaII restriction site close to the third CTCF-binding site (CTS3) within ICR1. Using bisulfite treatment and a real-time PCR-based methylation assay (QAMA), we analyzed the third and sixth CTCF-binding sites (CTS3, CTS6) in 5 patients with CTS3 hypomethylation, in 14 patients who were suspected to have SRS but were normal by Southern blot analysis, and in 1 patient with body asymmetry without any other features of SRS or Beckwith-Wiedemann syndrome (BWS). In all 5 patients with CTS3 hypomethylation, in 5 of 14 patients who were judged to be normal at CTS3 by Southern blot analysis and in the patient with isolated body asymmetry, we found CTS3 and CTS6 hypomethylation by QAMA. Using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), we obtained similar results at four additional ICR1 sites in the CTS6 region. These results show that ICR1 hypomethylation occurs more often in SRS patients than as previously thought as well as in isolated hemihypoplasia. Furthermore, we show that methylation analysis by QAMA and MLPA is more sensitive in detecting ICR1 hypomethylation than Southern blot analysis of CTS3.