Background: Genomic imprinting is required for normal development, and abnormal methylation of differentially methylated regions (iDMRs) controlling the parent of origin-dependent expression of the imprinted genes has been found in congenital disorders affecting growth, metabolism, neurobehavior, and in cancer. In most of these cases the cause of the imprinting abnormalities is unknown. Also, these studies have generally been performed on a limited number of CpGs, and a systematic investigation of iDMR methylation in the general population is lacking. Results: By analysing a vast number of either in-house generated or online available whole-genome methylation array datasets of unaffected individuals, and patients with complex and rare disorders, we determined the most common iDMR methylation profiles in a large population and identified many genetic and non-genetic factors contributing to their variability in blood DNA. We found that methylation variability was not homogeneous within the iDMRs and that the CpGs closer to the ZFP57 binding sites are less susceptible to methylation changes. We demonstrated the methylation polymorphism of three iDMRs and the atypical behaviour of several others, and reported the association of 25 disease- and 47 non-disease-complex traits as well as 15 Mendelian and chromosomal disorders with iDMR methylation changes. The most significantly associated complex traits included ageing, intracytoplasmic sperm injection, African versus European ancestry, female sex, pre- and postnatal exposure to pollutants and blood cell type compositions, while the associated genetic diseases included Down syndrome and the developmental disorders with molecular defects in the DNA methyltransferases DNMT1 and DNMT3B, H3K36 methyltransferase SETD2, chromatin remodelers SRCAP and SMARCA4 and transcription factor ADNP. Conclusions: These findings identify several genetic and non-genetic factors including new genes associated with genomic imprinting maintenance in humans, which may have a role in the aetiology of the diseases with imprinting abnormalities and have clear implications in molecular diagnostics.

Background A subset of individuals affected by imprinting disorders displays multi-locus imprinting disturbances (MLID). MLID has been associated with maternal-effect variants that alter the maintenance of methylation at germline-derived differentially methylated regions (gDMRs) in early embryogenesis. Pedigrees of individuals with MLID also include siblings with healthy phenotype. However, it is unknown if these healthy individuals have MLID themselves or if their methylation patterns differ from those associated with imprinting disorders, and in general, if MLID affects the clinical phenotype. Methods We have investigated gDMR methylation by locus-specific and whole-genome analyses in a family with multiple pregnancy losses, a child with Beckwith-Wiedemann syndrome (BWS) and a further child with no clinical diagnosis of imprinting disorder or other pathologies. Results We detected MLID with different methylation profiles in the BWS-affected and healthy siblings. Whole-exome sequencing demonstrated the presence of novel loss-of-function variants of NLRP5 in compound heterozygosity in the mother. The methylation profiles of the two siblings were compared with those of other cases with MLID and control groups by principal component analysis and unsupervised hierarchical clustering, but while their patterns were clearly separated from those of controls, we were unable to cluster those associated with specific clinical phenotypes among the MLID cases. Conclusion The identification of two novel maternal-effect variants of NLRP5 associated with poly-abortivity and MLID adds further evidence to the role of this gene in the maintenance of genomic imprinting in early embryos. Furthermore, our results demonstrate that within these pedigrees, MLID can also be present in the progeny with healthy phenotype, indicating that some sort of compensation occurs between altered imprinted loci in these individuals. The analysis of larger cohorts of patients with MLID is needed to formulate more accurate epigenotype-phenotype correlations.