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Beckwith-Wiedemann syndrome (BWS) is a rare disorder characterized by overgrowth and predisposition to embryonal tumors. BWS is caused by various epigenetic and/or genetic alterations that dysregulate the imprinted genes on chromosome region 11p15.5. Molecular analysis is required to reinforce the clinical diagnosis of BWS and to identify BWS patients with cancer susceptibility. This is particularly crucial prenatally because most signs of BWS cannot be recognized in utero.We established a reliable molecular assay by pyrosequencing to quantitatively evaluate the methylation profiles of ICR1 and ICR2. We explored epigenotype-phenotype correlations in 19 patients that fulfilled the clinical diagnostic criteria for BWS, 22 patients with suspected BWS, and three fetuses with omphalocele. Abnormal methylation was observed in one prenatal case and 19 postnatal cases, including seven suspected BWS. Seven cases showed ICR1 hypermethylation, five cases showed ICR2 hypomethylation, and eight cases showed abnormal methylation of ICR1 and ICR2 indicating paternal uniparental disomy (UPD). More cases of ICR1 alterations and UPD were found than expected. This is likely due to the sensitivity of this approach, which can detect slight deviations in methylation from normal levels. There was a significant correlation (p < 0.001) between the percentage of ICR1 methylation and BWS features: severe hypermethylation (range: 75–86%) was associated with macroglossia, macrosomia, and visceromegaly, whereas mild hypermethylation (range: 55–59%) was associated with umbilical hernia and diastasis recti. Evaluation of ICR1 and ICR2 methylation by pyrosequencing in BWS can improve epigenotype-phenotype correlations, detection of methylation alterations in suspected cases, and identification of UPD.

Genomic imprinting, resulting in parent-of-origin-dependent gene expression, is mainly achieved by DNA methylation. IGF2and H19, belonging to the same cluster of imprinted genes and regulated by ICR1, DMR2 and H19promoter elements, play a major role in fetal/placental growth. Using quantitative approaches, we explored the epigenetic modulation of IGF2/H19during human development in 60 normal and 66 idiopathic IUGR (Intrauterine Growth Restriction) pregnancies, studying embryonic (cord blood) and extraembryonic (placenta and umbilical cord) tissues. We found ICR1 normal methylation levels (~50%) and H19 promoter/DMR2 hypomethylation in extra-embryonic tissues. In contrast, in embryonic samples the three loci displayed normal methylation values comparable to those in postnatal blood. This feature is stably maintained throughout gestation and does not vary in IUGR cases. We reported asymmetric allelic expression of H19and IGF2 as a common feature in pre- and post-natal tissues, independent of H19 promoter and DMR2 methylation levels. In addition, we excluded in IUGR posttranscriptional IGF2 interference possibly related to miRNA 483-3p (IGF2, intron 2) expression defects. Through LINE1 methylation analysis, we observed a methylation gradient with increasing methylation from pre- to post-natal life. The involvement of UPD (Uniparental Disomy) in IUGR aetiology was excluded. Our data indicate that: i) ICR1 3 methylation status is a necessary and sufficient condition to drive the imprinting of IGF2and H19present in embryonic as well as in extra-embryonic tissues; ii) hypomethylation of H19 promoter and DMR2 does not influence the expression pattern of IGF2and H19; iii) there is a gradient of global methylation, increasing from extra-embryonic to embryonic and adult tissues. Finally, because of placental hypomethylation, cautions should be exercised in diagnosis of imprinting diseases using chorionic villi.