Lack of involvement of known DNA methyltransferases in familial hydatidiform mole implies the involvement of other factors in establishment of imprinting in the human female germline

Abstract

Background: Differential methylation of the two alleles is a hallmark of imprinted genes. Correspondingly, loss of DNA methyltransferase function results in aberrant imprinting and abnormal post-fertilization development. In the mouse, mutations of the oocyte-specific isoform of the DNA methyltransferase Dnmt1 (Dnmt1o) and of the methyltransferase-like Dnmt3L gene result in specific failures of imprint establishment or maintenance, at multiple loci. We have previously shown in humans that an analogous inherited failure to establish imprinting at multiple loci in the female germline underlies a rare phenotype of recurrent hydatidiform mole.

Results: We have identified a human homologue of the murine Dnmt1o and assessed its pattern of expression. Human DNMT1o mRNA is detectable in mature oocytes and early fertilized embryos but not in any somatic tissues analysed. The somatic isoform of DNMT1 mRNA, in contrast, is not detectable in human oocytes. In the previously-described family with multi-locus imprinting failure, mutation of DNMT1o and of the other known members of this gene family has been excluded.

Conclusions: Mutation of the known DNMT genes does not underlie familial hydatidiform mole, at least in the family under study. This suggests that trans-acting factors other than the known methyltransferases are required for imprint establishment in humans, a concept that has indirect support from recent biochemical studies of DNMT3L.

Figure 1

a. PIP (percentage identity plot) diagram for human and murine DNMT1. The horizontal axis represents the human genomic sequence and has been annotated to show the position of the novel exon 1o, which unlike the previously recognized DNMT1 exons, shows no sequence conservation relative to mouse. Tall black rectangles indicate the positions of the known human DNMT1 exons. Low grey and white rectangles indicate regions with CpG-island like features (CpG/GpC respectively ≥ 0.75 and ≥ 0.60). b. Alternative first exon (1o) of human DNMT1. The exon is in upper case. Underlining indicates the PCR primers used for genomic sequence analysis (flanking) and double underlining for RT-PCR analysis of oocyte material. c. Detection of DNMT1 expression in cDNA from human oocytes and embryos. The panel labelled DNMT1o shows PCR reactions using the upstream primer indicated in panel b, unique to the novel isoform of human DNMT1. In the DNMT1s panel, an upstream primer in the previously defined exon 1 was used. The ZP3 panel shows control reactions that detect the mRNA for zona pellucida protein ZP3, which is expressed at all stages of oogenesis. Lanes 1–2, primordial follicles (P); 3, mix of primordial and early primary; 4, mix of early primary and primary (1°); 5, primary; 6, secondary (2°); 7–9, ovulated oocytes; 10, 2-cell embryos (2); 11–12, 4-cell embryos (4); 13–16, blastocysts; 17, negative control (0).