Epigenetic defects (demethylation of KCNQ1OT1
or hypermethylation of H19
) account for about 70% of molecularly proved cases of BWS. Some of these cases display a microdeletion of the centromeric18
imprinting centres of the 11p15 region. Recent reports3,4,5,6,11
have highlighted an increase in the risk of BWS in children born after the use of ART. The molecular defect in patients with BWS conceived by ART consists of demethylation of the KvDMR1/KCNQ1OT1
locus, suggesting that ART impairs the acquisition (during oocyte maturation) or maintenance (after fertilisation) of the maternal methylation marks of the centromeric imprinting centre of the 11p15 region. The timing of methylation mark acquisition by maternal imprinted genes during oocyte maturation has been studied only in mice, in which it was found to be asynchronous.9
However, the timing of KvDMR1/KCNQ1OT1
methylation in mice is unknown. Little is known about the acquisition of methylation marks in humans, but the studies carried out to date have suggested that various key aspects of the timing of DNA methylation acquisition and maintenance are conserved in the germ line and early embryo.21,22,23
Thus, maternal methylation marks are probably acquired sequentially in human oocytes, as in mouse oocytes.
The methylation status of imprinted loci other than the 11p15 region has not been investigated in patients with BWS. In particular, no previous study has attempted to determine whether the epigenetic imprinting error after the use of ART is restricted to a specific imprinted domain or randomly distributed. In this study, we compared methylation status at various imprinted loci in patients with BWS born after the use of ART and in patients with the same epigenetic defect (ie, demethylation of KCNQ1OT1) conceived naturally.
We found that some patients with BWS conceived by ART displayed abnormal methylation at loci other than 11p15. Analysis of the ART procedures used in the three patients with abnormal methylation of several loci implicated no specific ART procedure. The methylation defect involved imprinted loci and the methylation pattern of constitutive heterochromatin was normal in patients with BWS conceived by ART. Some naturally conceived patients with BWS also displayed abnormal methylation at loci other than 11p15. It indicates that ART procedures are not specifically involved in loss of methylation at various imprinted loci.
The loss of methylation at the various imprinted loci was only partial, suggesting mosaicism of the epimutation. This mosaic pattern suggests that the epigenetic error occurs after fertilisation, during early development, rather than during oocyte maturation. Similar results were observed in mice.24
There are therefore two possibilities:
- the mechanism involved in the epigenetic error is different in patients with a methylation defect involving various loci and in patients with a defect restricted to KCNQ1OT1; or
- the mechanism is the same in these two sets of patients, but the degree of expression is different, ranging from demethylation of the most vulnerable locus (KCNQ1OT1) to the demethylation of several imprinted loci.
Evidence in favour of this second possibility is provided by the observation of variable levels of demethylation in people displaying a loss of methylation at various loci (table 1). The high incidence of monozygotic twinning with the affected twin displaying a loss of methylation at KCNQ1OT112,13
suggests that the KCNQ1OT1
locus is particularly vulnerable to epigenetic errors during early development.25
The mechanisms safeguarding imprinting marks during the wave of demethylation that occurs during early pre‐implantation development remain poorly characterised. DNA methyltransferases and methyl‐binding domain proteins are probably key regulators in this process, and a model based on errors in trafficking of the oocyte isoform of DNMT1 has been proposed to explain the genetics of BWS in monozygotic twins.25
Recently, Arnaud et al26
showed that a maternal imprint can be acquired in the absence of Dnmt3L in female germ cells. This incomplete penetrance of Dnmt3L deficiency was neither locus nor embryo specific, but stochastic suggesting that in the absence of Dnmt3L other factors can mark individual DMRs.
Surprisingly, patients with defects at loci other than 11p15 do not display other phenotypes. However, the slight difference in the frequency of macrosomia, although non‐significant, could be accounted for by the involvement of other loci. PEG1/MEST
is believed to be involved in fetal growth, although its precise function is unknown. Disruption of the PEG1/MEST
gene causes embryonic growth retardation when paternally transmitted.27
A loss of maternal methylation marks at this locus may therefore result in the biallelic expression of PEG1/MEST
and fetal overgrowth. Regarding the IGF2R
gene, differential methylation of maternal and paternal DMR2 region is maintained in humans28
and some humans do appear to display transcriptional imprinting of IGF2R
IGF2R is responsible for the clearance and inactivation of IGF2. A loss of maternal IGF2R
/DMR2 methylation would probably decrease IGF2R
expression, thereby increasing IGF2 availability and fetal growth.
Recently, some patients with a clinical diagnosis of transient neonatal diabetes mellitus (MIM 601410) caused by loss of methylation at the transient neonatal diabetes mellitus maternally methylated imprinted domain on 6q24 were described with a partial loss of methylation at KCNQ1OT1
As in our series, the phenotype of patients with involvement of both loci (6q24 and 11p15) was not very different from the phenotype of patients with the involvement of only 6q24.31
In this study, we searched for imprinting defects involving loci other than the KCNQ1OT1 locus in patients born after the use of ART. Our original hypothesis that other loci are involved only in patients conceived by ART was not confirmed. Instead, we showed the involvement of other imprinted loci in some naturally conceived patients with BWS. Moreover, the pattern of cellular mosaicism observed suggests that the imprinting defect occurs after fertilisation via a mechanism impairing the maintenance of maternal imprints. The mechanisms controlling the protection of imprinted loci against demethylation remain unclear, but our data suggest that this protection fails in these patients, resulting in a loss of maternal imprints.