The present study was designed to determine whether imprinting defects contribute to SGA in ICSI children. The patients were recruited from a prospective study on the outcome of ICSI.11
Among 19 ICSI/SGA children, we identified only one child with putative imprinting defects of KCNQ1OT1
. None of the children had Silver–Russell syndrome, which often results from hypomethylation of IGF2/H19.8
Our results show that imprinting defects are not frequent in ICSI/SGA children, at least not at the six loci investigated in this study. However, our study is limited by the fact that we could investigate only one tissue (buccal smear) at only one time point. Most importantly, we did not have newborn and placental material. Owing to the fact that imprinting defects are often mosaic (see discussion below), the frequency of imprinting defects might actually be higher than observed in our study.
By SeQMA, one child had 72–74% methylation of KCNQ1OT1 and 60–63% methylation of PEG1. The parents were normal at these loci. The latter finding and our genomic sequence analysis exclude the possibility that our analysis was confounded by an SNP, and indicate that the child has a de novo hypermethylation. Although the results were confirmed by an independent method (COBRA), the findings in PEG1 are borderline. We note, however, that child no. 33 has the highest degree of PEG1 methylation among all 48 children. The results between two SeQMA experiments are reassuringly similar, showing that there is little inter-experiment variation, but there appears to be a certain degree of inter-individual variation in methylation, which makes the detection of mosaic imprinting defects difficult.
Do our findings make sense? Hypermethylation of KCNQ1OT1
means that the paternal allele is methylated, which is normally unmethylated. PEG1
is located in 7q32, encodes a member of the alpha/beta hydroxylase fold family and is paternally expressed in human fetal tissues. The promoter region is methylated on the maternal chromosome and unmethylated on the paternal chromosome.15
Maternal uniparental disomy 7 (complete methylation at this locus) is associated with pre- and postnatal growth retardation, but the role of PEG1
in this condition is unknown. Lefebvre et al16
showed that heterozygous mice that inherited a mutant allele from the paternal germ line were smaller and lighter, but otherwise fertile.
is located in 11p15, encodes the cyclin-dependent kinase inhibitor p57KIP2 and is maternally expressed. Regulation of imprinted CDKN1C
expression is regulated by the maternally methylated KvDMR1
within intron 10 of the KCNQ1
gene, which also regulates expression of the paternally expressed KCNQ1OT1
Loss of CDKN1C
expression is associated with overgrowth (Beckwith–Wiedemann syndrome5
). Maternal duplication of 11p15 is associated with growth retardation,18, 19
although this effect could also involve the IGF2/H19
cluster. Up to now hypermethylation of the KCNQ1OT1
locus has not been reported in humans, but probably leads to overexpression of CDKN1C
and thus may cause fetal growth retardation. A study of Fitzpatrick et al20
demonstrated that a paternal inheritance of a deletion of Kvdmr1
results in overexpression of six maternally expressed genes, including Cdkn1c
. The main feature of these knockout mice is growth retardation.
In summary, we note that the methylation changes of KCNQ1OT1 and PEG1 in child no. 33 are in the expected direction.
Assuming the methylation changes are authentic, what might be the cause? In general, epimutations after assisted reproduction may be linked to the fertility problems of the parents (father or mother) or the procedure. On the basis of studies in patients with Angelman syndrome,21, 22
Beckwith–Wiedemann syndrome23, 24, 25, 26
and animal models,6
it has been suggested that superovulation13, 27
or cell culture23, 24
might affect methylation at certain imprinted loci. These procedures are unlikely to be the reason for the abnormal methylation pattern in child no. 33. In the aforementioned studies, loss of methylation at maternal alleles at the SNRPN
loci had been found. In child no. 33, we have found hypermethylation of the paternal allele of KCNQ1OT1
and possibly PEG1
A study of Kobayashi et al28
revealed that sperm from infertile men carry a higher risk of transmitting incorrect primary imprints to their offspring. Moreover, they found out that errors were more frequent at maternally methylated DMRs in the ejaculated sperm than at paternally methylated DMRs. Aberrant methylation of several maternally methylated loci such as PEG1
in the ejaculated sperm are probably the result of errors in imprint erasure. Transmission of the aberrant imprint might thus be promoted during male infertility treatment.28, 29, 30
Therefore, it is likely that a sperm of the affected child′s father, who had oligozoospermia, carried an incorrect imprint (hypermethylation at KCNQ1OT1
), which was transmitted to the child.
Interestingly, Kagami et al31
found partial hypermethylation of PEG1
in a patient with Silver–Russell syndrome conceived after in vitro
fertilization. Furthermore, four of the eight abnormally methylated cytosines were also methylated in the father. It was inferred, therefore, that the paternal PEG1
allele with mildly hypermethylated DMR was further methylated and transmitted to the patient.
If child no. 33 was conceived from a sperm carrying methylated KCNQ1OT1 and PEG1 alleles, why does she not have 100% methylation at these loci? Partial hypermethylation of these loci in child no. 33 reflects a mosaic situation, that is, the presence of hypermethylated cells and normal cells. Mosaic hypermethylation may result (i) from the postzygotic gain of methylation at these loci, possibly as a failure of a cell to protect the unmethylated paternal alleles against de novo methylation or (ii) from the postzygotic loss of an aberrant methylation imprint. It is difficult to link these defects to the assisted reproduction technology procedure.
On the basis of the methylation changes found in sperm of infertile men, including the PEG1
locus, it is tempting to speculate that the methylation changes in child no. 33 are linked to the fertility problem of the father. The following scenario would be compatible with the published data and our findings. Child no. 33 was conceived from a sperm that was abnormally methylated at KCNQ1OT1
, probably as a result from a failure to erase the maternal methylation imprint during spermatogenesis. It has been shown in patients with Prader–Willi syndrome and a sporadic imprinting defect, that the chromosome carrying the imprint defect is always inherited from the paternal grandmother,32
strongly suggesting that defects in imprint erasure do occur. In contrast to a real maternal methylation imprint, the aberrant methylation pattern may not include each CpG within the DMR. As a consequence, this abnormal imprint may be less stable during postzygotic development, leading to complete loss in some cells. As such cells would have a proliferative advantage relative to a cell with KCNQ1OT1
hypermethylation, this cell line would outgrow the affected cell line. This scenario is speculative, but would explain the catch-up growth in this child.
In summary, imprinting defects appear to be a rare finding in ICSI children born SGA, at least at the loci investigated in this study. To the best of our knowledge, hypermethylation of KCNQ1OT1 has never been observed before in a normal or a growth-retarded child. It may represent a rare stochastic event or be linked to paternal subfertility, but not to in vitro fertilization/ICSI. Given the rare frequency of imprinting defects and the instability of defective imprints at certain loci, it will be necessary to collect newborn and placental tissue and to examine more children.