Abstract
BackgroundMaternal effect mutations in the components of the subcortical maternal complex (SCMC) of the human oocyte can cause early embryonic failure, gestational abnormalities and recurrent pregnancy loss. Enigmatically, they are also associated with DNA methylation abnormalities at imprinted genes in conceptuses: in the devastating gestational abnormality biparental complete hydatidiform mole (BiCHM) or in multi-locus imprinting disease (MLID). However, the developmental timing, genomic extent and mechanistic basis of these imprinting defects are unknown. The rarity of these disorders and the possibility that methylation defects originate in oocytes have made these questions very challenging to address.MethodsSingle-cell bisulphite sequencing (scBS-seq) was used to assess methylation in oocytes from a patient with BiCHM identified to be homozygous for an inactivating mutation in the human SCMC component KHDC3L. Genome-wide methylation analysis of a preimplantation embryo and molar tissue from the same patient was also performed.ResultsHigh-coverage scBS-seq libraries were obtained from five KHDC3Lc.1A>G oocytes, which revealed a genome-wide deficit of DNA methylation compared with normal human oocytes. Importantly, germline differentially methylated regions (gDMRs) of imprinted genes were affected similarly to other sequence features that normally become methylated in oocytes, indicating no selectivity towards imprinted genes. A range of methylation losses was observed across genomic features, including gDMRs, indicating variable sensitivity to defects in the SCMC. Genome-wide analysis of a pre-implantation embryo and molar tissue from the same patient showed that following fertilisation methylation defects at imprinted genes persist, while most non-imprinted regions of the genome recover near-normal methylation post-implantation.ConclusionsWe show for the first time that the integrity of the SCMC is essential for de novo methylation in the female germline. These findings have important implications for understanding the role of the SCMC in DNA methylation and for the origin of imprinting defects, for counselling affected families, and will help inform future therapeutic approaches.
Highlights
Maternal effect mutations in the components of the subcortical maternal complex (SCMC) of the human oocyte can cause early embryonic failure, gestational abnormalities and recurrent pregnancy loss
Methylation defects in biparental complete hydatidiform mole caused by KHDC3L mutation Patient D, a 27-year-old woman homozygous for a confirmed loss-of-function mutation (A to G at the + 1 position in start codon) in KHDC3L with multiple consanguineous marriages in her extended family (Additional file 3: Figure S1A,B), had a history of 2 BiCHMs
For the purpose of this and subsequent analyses, maternal germline differentially methylated regions (gDMRs) are defined as DMRs methylated in oocytes but not in sperm and associated with known imprinted genes (32 features); paternal gDMRs as DMRs methylated in sperm but not oocytes and associated with known imprinted genes (1 feature); placenta-specific gDMRs as CpG islands (CGIs) methylated in oocytes but not sperm and retaining allelic differential methylation selectively in placenta (15 features [13];); and secondary gDMRs as elements in known imprinted loci that acquire allelic differential methylation after fertilisation (14 features)
Summary
Maternal effect mutations in the components of the subcortical maternal complex (SCMC) of the human oocyte can cause early embryonic failure, gestational abnormalities and recurrent pregnancy loss They are associated with DNA methylation abnormalities at imprinted genes in conceptuses: in the devastating gestational abnormality biparental complete hydatidiform mole (BiCHM) or in multi-locus imprinting disease (MLID). Amongst the key outstanding questions are (1) whether there is a defective establishment of methylation in oocytes or a failure to maintain methylation at imprinted loci in pre-implantation embryos during genome-wide erasure of gametic methylation and (2) whether LoM is limited to imprinted loci or is more widespread All these possibilities could pertain because, in addition to undergoing genome-wide de novo methylation [18], the oocyte provides the key factors required for the complex methylation reprogramming events in the cleavage-stage embryo, including gDMR methylation maintenance [19, 20]. This is the first study to assess the origin of an imprinting disorder in the human germline, demonstrating the importance of the integrity of the SCMC for DNA methylation establishment in the oocyte
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