In this cohort of patients referred for investigation of RSS/unexplained growth restriction, 37% (29 of 79) of DNA samples showed LOM at one or more imprinted loci. LOM at H19
was the most common methylation anomaly in such patients. Most other studies have concluded that LOM at H19
is very unlikely in those patients without features of RSS;7, 8, 15, 16
however, in this series two patients with LOM at H19
did not have a diagnosis of RSS clearly suggested on the referral details. They were both from the anonymised recruitment arm and therefore detailed phenotypic information has not been confirmed, but both were referred by clinical geneticists. In one of these patients there was a complex clinical picture and multiple congenital anomalies. It is possible that RSS had not been suspected clinically, because of the severity of comorbid features, or his young age.
We found HIL in 2 of 23 patients with LOM at H19
, a phenomenon which was not observed in a previous study that tested a smaller number of DMRs.14
These two patients are remarkable. Patient 1 had a half-sibling with BWS. Siblings with RSS and BWS due to underlying HIL have not been previously described, and this family probably represents a new genetic disorder. Patient 2 was one of discordant monozygous twins, which might be aetiologically relevant: the incidence of monozygotic twinning in BWS is increased20, 21
and some of these show HIL.22
Monozygous twins discordant for RSS have also been reported ;23, 24, 25
although, to our knowledge this latter group has not been investigated for HIL. We believe the discovery of HIL in patient 2 with growth restriction may represent a new aetiological group, with HIL arising as part of the twinning process.
With the exception of patient 2, who had LOM at GRB10
on chromosome 7 as part of a wider HIL, we did not detect epigenetic changes on chromosome 7 to account for the restricted growth in this cohort. These results corroborate those of another study that identified no methylation change at MEST/PEG1
in 54 RSS patients.26
Taken with our own results this suggests that isolated imprinting abnormalities of MEST1/PEG1, GRB10
on chromosome 7 are not a common cause of growth retardation or RSS.
Surprisingly, this study has also revealed that LOM at KCNQ1OT1,
previously associated with BWS, can be found in patients with growth restriction (patients 2 and 5). Recently there has been another report of LOM at KCNQ1OT1
in three children without clinical BWS.27
In our study, although MLPA did not reveal a copy number change in the 11p15 region, we cannot exclude an atypical paternal duplication, or a maternal deletion; however, it is difficult to explain the phenotype of growth restriction in the context of the epigenetic result in these two patients. In patient 2, the HIL involving multiple loci, including H19,
may have prevented a net overgrowth; yet, a similar pattern, with hypomethylation at both maternally and paternally methylated loci, was also seen in discordant twins with BWS.22
The modest changes in methylation at multiple loci in patient 2 may reflect a dilutional effect resulting from circulation sharing with her twin. For patient 5, we did not show HIL, though we cannot exclude hypomethylation of DMRs not analysed in this study.
The finding of a complete gain of methylation at IGF2R in 7 of 79 patients is also intriguing. This occurred significantly more commonly in the patients (cases) than in our control group (P=0.002 using Fisher's Exact test).
Although the proliferative effects of IGF2 are mediated through the IGF1 receptor (IGF1R), the main function of the IGF2 receptor is anti-proliferative, achieved by clearing IGF2 from the circulation.28
Not surprisingly therefore, Igf2r
knockout mice are up to 30% larger than wild-type mice.29, 30, 31
The expression of human and mouse IGF2R
differs: in mice, Igf2r
imprinted expression is observed in all fetal and adult tissues; in humans, IGF2R
imprinted expression has not been found in adult tissues, but has been found in fetal tissues and Wilms' tumours, in a proportion of samples tested.32
There is also a difference in the DMRs between these two species: DMR1, in the promoter of the Igf2r
gene, occurs only in mice; DMR2 occurs in both species and is located in intron 2, methylated on the maternal allele. In mice the DMR2 acts as a promoter for Air
, a non-coding RNA, which is antisense to Igf2r
. Maternal methylation of DMR2 silences maternal Air
, allowing monoallelic maternal expression of IGF2R
. There is now some evidence of a human Air homologue.33
Given this control and expression pattern, we hypothesised that a gain of methylation at DMR2 in humans might increase the expression of IGF2R
(at a critical stage of embryonic development), which could lead to a decrease in circulating IGF2, and so decreased growth. The opposite epigenetic modification, in the form of demethylation of the DMR2 of Igf2r,
leads to overgrowth of sheep.34
Furthermore, in humans a partial demethylation of DMR2 was detected in one of 55 patients with idiopathic overgrowth, and was associated with a decreased serum IGF2R level and decreased IGF2 binding, at age 3 years.35
We are not able to go back to the patients in this study to compare clinical findings in this subgroup with the rest of the cohort, or test IGF2 levels. The available clinical details reveal failure to thrive and post natal growth retardation, with or without prenatal growth retardation (). The findings at IGF2R
warrant further investigation and may have highlighted a novel subset of growth retarded individuals.
The main limitation of this study is the minimal phenotypic information available on the 60 individuals within the anonymised cohort. It is recognised that referral cards can be difficult to interpret and this may limit our epigenotype–phenotype correlations; however, almost half of this group originated from Clinical Geneticists who may provide more reliable referral details. Furthermore, this group does reflect routine clinical practice, being drawn from unselected referrals to a service laboratory of patients with growth restriction. Thus, this study mirrors the real difficulty of diagnosing RSS in the clinic: in many instances, although doctors know there is definite intrauterine growth retardation and or short stature, there maybe uncertainty about the presence of other RSS features, some of which evolve with time.
We believe this is the most extensive molecular study of its kind, analysing 11 DMRs in individuals with suspected RSS or growth retardation. We report new cases of HIL in patients with LOM at H19. We have ascertained further patients with LOM at KCNQ1OT1 who do not have clinical BWS. Although we did not identify the loci on chromosome 7 that may account for the growth restriction observed in maternal UPD7, we did identify a potentially important change involving IGF2R. We believe this study warrants confirmation prospectively alongside accurate clinical phenotyping.