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The identification of multilocus imprinting disturbances (MLID) appears fundamental to uncover molecular pathways underlying imprinting disorders (IDs) and to complete clinical diagnosis of patients. However, MLID genetic associated mechanisms remain largely unknown. To characterize MLID in Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, we profiled by MassARRAY the methylation of 12 imprinted differentially methylated regions (iDMRs) in 21 BWS and 7 SRS patients with chromosome 11p15.5 epimutations. MLID was identified in 50% of BWS and 29% of SRS patients as a maternal hypomethylation syndrome. By next-generation sequencing, we searched for putative MLID-causative mutations in genes involved in methylation establishment/maintenance and found two novel missense mutations possibly causative of MLID: one in NLRP2, affecting ADP binding and protein activity, and one in ZFP42, likely leading to loss of DNA binding specificity. Both variants were paternally inherited. In silico protein modelling allowed to define the functional effect of these mutations. We found that MLID is very frequent in BWS/SRS. In addition, since MLID-BWS patients in our cohort show a peculiar pattern of BWS-associated clinical signs, MLID test could be important for a comprehensive clinical assessment. Finally, we highlighted the possible involvement of ZFP42 variants in MLID development and confirmed NLRP2 as causative locus in BWS-MLID.

Genomic imprinting, resulting in parent-of-origin-dependent gene expression, is mainly achieved by DNA methylation. IGF2 and H19, belonging to the same cluster of imprinted genes and regulated by ICR1, DMR2 and H19 promoter elements, play a major role in fetal/placental growth. Using quantitative approaches, we explored the epigenetic modulation of IGF2/H19 during 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 ( approximately 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 H19 and 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 post-transcriptional 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: (1) ICR1 methylation status is a necessary and sufficient condition to drive the imprinting of IGF2 and H19 present in embryonic as well as in extra-embryonic tissues; (2) hypomethylation of H19 promoter and DMR2 does not influence the expression pattern of IGF2 and H19; (3) 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.