Introduction

Genomic imprinting is an epigenetic phenomenon that leads to parent-specific differential expression of a subset of mammalian genes. So far, >100 imprinted genes have been identified in humans and mice, and most imprinted genes often form clusters, or imprinting domains. The expression of imprinted genes within these domains is regulated by imprinting control regions (ICRs).1, 2 ICRs are identical to differentially methylated regions (DMRs), which are characterized by DNA methylation on one of the two parental alleles, or maternally methylated DMRs and paternally methylated DMRs. In addition, there are two classes of imprinted DMRs, gametic DMRs and somatic DMRs. Gametic DMRs acquire DNA methylation during gametogenesis, and the methylation is maintained from zygote to somatic cells during all developmental stages. Most gametic DMRs are identical to ICRs. Methylations of somatic DMRs are established during early embryogenesis after fertilization under the control of nearby ICRs.1, 2

As most imprinted genes have an important role in the growth and development of embryos, placental formation, and metabolism, aberrant expression of imprinted genes due to epigenetic or genetic abnormalities is often implicated in the pathogenesis of human disorders such as congenital anomalies and tumors.1, 2 Epigenetic abnormality leading to aberrant expression of imprinted genes mostly includes aberrant hypomethylation or hypermethylation at ICRs. Genetic abnormalities include uniparental disomies, chromosomal deletions, duplications, translocations, inversions of imprinting domains, and point mutations of imprinted genes. Representative imprinting disorders and their corresponding imprinted loci are as follows: Beckwith–Wiedemann syndrome (BWS) at 11p15.5, Prader-Willi/Angelman syndromes at 15q11-q13, pseudoparahypothyroidism type 1b at 20q13.3, Silver–Russell syndrome (SRS) at 11p15.5 and chromosome 7, and transient neonatal diabetes mellitus type 1 at 6q24.

Here, we review BWS, focusing especially on imprinting mechanisms of 11p15.5, epigenetic and genetic etiologies leading to aberrant expression of corresponding imprinted genes, relationships between epigenetic/genetic alterations and clinical features, and associated childhood tumors. We also describe the relationship between assisted reproductive technology (ART) and imprinting disorders and explore multilocus hypomethylation disorders (MHDs).

Clinical features and causative alterations of BWS

BWS (OMIM #130650) is a pediatric overgrowth disorder that is characterized by the peculiar traits of prenatal and postnatal macrosomia, macroglossia, abdominal wall defects as originally described by Beckwith and Wiedemann.3, 4 The incidence has been reported to be 1 in 13 700,5 and the male-to-female ratio is ∼1:1. BWS also shows other variable features, including anterior ear lobe creases and/or posterior helical pits, neonatal hypoglycemia, intra-abdominal visceromegaly, cytomegaly of adrenal fetal cortex, renal abnormalities, hemihyperplasia and cleft palate. The development of embryonal tumors (for example, Wilms’ tumor, hepatoblastoma and rhabdomyosarcoma) is an important feature of BWS, and the overall tumor risk has been estimated at 7.5% with a range of 4–21%.6, 7 Although several clinical criteria have been proposed so far,8, 9, 10 there is no single unified criterion. However, a criteria scheme proposed by Weksberg et al.11 is generally accepted for clinical diagnosis: the presence of at least three major findings, or two major findings and one minor finding, from those reported in Table 1. Simpson–Golabi–Behmel syndrome, Costello syndrome, Perlman syndrome, Sotos syndrome and mucopolysaccharidosis VI (Maroteaux–Lamy syndrome) are considered as differential diagnoses.

Table 1 Major and minor finings associated with Beckwith–Wiedemann syndrome11
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Approximately 85% of BWS cases are sporadic; the other 15% are familial showing autosomal dominant inheritance. The relevant imprinted chromosomal region in BWS, 11p15.5, consists of two independent imprinting domains, IGF2/H19 and CDKN1C/KCNQ1OT1. Several causative alterations have been identified for sporadic cases of BWS: loss of methylation (LOM) at KvDMR1 (∼50%), gain of methylation (GOM) at H19DMR (∼5%), paternal uniparental disomy (patUPD; ∼20%), CDKN1C mutations (∼5%), duplications of 11p15 (<1%) and translocations or inversions involving 11p15 (<1%) (Table 2).11, 12, 13 However, no alteration of 11p15.5 can be found for ∼20% of BWS cases. Interestingly, among these causative alterations, methylation abnormalities, such as KvDMR1-LOM and H19DMR-GOM, and patUPD are mosaic in the patients; however, other genetic alterations including CDKN1C mutation are essentially not mosaic.

Table 2 Correlation between epigenetic/genetic alteration and clinical features
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Imprinting mechanisms of 11p15.5 and etiologies of BWS

The IGF2/H19 domain

The important genes in this domain are insulin-like growth factor 2 (IGF2) and H19. IGF2 is expressed from the paternal allele, and the gene product has an important role in development and growth, whereas H19 is a maternally-expressed, non-coding RNA, which may function as a tumor suppressor, but whose precise biological role remains unresolved.14, 15 One study reported that H19 is a miRNA precursor expressed in human keratinocytes and neonatal mice, suggesting its involvement during development.16 The ICR of this domain is H19DMR, which is located 2 kb upstream of H19 and is methylated on the paternal but not the maternal allele (Figure 1). The methylation of H19DMR is established during spermatogenesis.17, 18 This ICR, which contains seven CCCTC-binding factor (CTCF) binding sites in human and four in mouse, regulates the reciprocal expression of IGF2 and H19 by functioning as a chromatin insulator. On the maternal allele, CTCF binding at the insulator elements within unmethylated H19DMR blocks enhancers downstream of H19 from accessing IGF2 promoters. On the paternal allele, as the methylation of H19DMR prevents CTCF binding, the enhancers can access IGF2 promoters.19, 20 Thus, these mechanisms lead to paternal expression of IGF2 and maternal expression of H19. Recent chromatin conformation studies showed that CTCF binding at regulatory regions other than H19DMR and the enhancers surrounding the domain formed allele-specific chromatin loops, depending on the methylation of H19DMR, in order to regulate the expression of IGF2 and H19. For these CTCF-dependent chromatin loop formations, the recruitment of cohesin to CTCF-binding sites is required and cohesin stabilizes the chromatin conformations.21, 22

Figure 1
figure 1

Imprinting domains at 11p15.5. Upper panel indicates the imprinting mechanisms in normal individuals. As for the IGF2/H19 domain, the insulator model is shown. On the maternal chromosome, the binding of CTCF to unmethylated H19DMR blocks enhancers from accessing IGF2 promoters. In contrast, on the paternal allele, as the methylation of H19DMR prevents CTCF binding, the enhancers can access IGF2 promoters. Thus, these mechanisms lead to paternal expression of IGF2 and maternal expression of H19. Please refer to the text for the chromatin loop model. As for the CDKN1C/KCNQ1OT1 domain, on the paternal chromosome, it has been proposed that CDKN1C is repressed by KCNQ1OT1 RNA coating and by a silencer and an insulator near the KvDMR1, which is likely regulated by CTCF. A putative enhancer within the KCNQ1 locus acts on maternal expression of CDKN1C. The lower panel displays causative alterations of BWS. Vertical arrows with maternal translocations and inversions indicate chromosomal break points. Blue: paternal expressed genes; red: maternal expressed genes; green diamond: enhancers (putative enhancer in CDKN1C/KCNQ1OT1 domain); wavy line: non-coding RNA transcribed from the paternal KCNQ1OT1 gene.

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In ∼5% of BWS patients, gain of DNA methylation occurs on the normally unmethylated maternal H19DMR (H19DMR-GOM) (Figure 1, Table 2). Aberrant DNA methylation at maternal H19DMR is accompanied by a change of histone modification from accessible H3K9ac and bivalent H3K4me2/H3K27me3 to repressive H3K9me3 and H4K20me3.22 The aberrant DNA methylation prevents CTCF binding to maternal H19DMR, and the chromatin loop formation changes from maternal-type to paternal-type due to aberrant DNA methylation and histone modification change. The chromatin conformation change drags the enhancers into the vicinity of IGF2, leading to biallelic expression and loss of imprinting of IGF2 and reduced expression of H19. Overexpression of IGF2 and reduced expression of H19 induce the BWS phenotype. One representative phenotype of H19DMR-GOM is hemihyperplasia (Table 2).12

The majority of GOM cases show an isolated epigenetic alteration; however, ∼20% of GOM cases are associated with genetic alterations, which are variable length microdeletions including CTCF-binding sites and point mutations and a deletion at the octamer-binding protein (OCT) binding site.23 These genetic alterations lead to maternal H19DMR not being able to maintain an unmethylated status.24, 25 However, the mechanism by which isolated H19DMR-GOM occurs is still unknown. As a certain number of cases with isolated H19DMR-GOM show variable hypermethylation, patients have an epigenetic mosaic of normal cells and aberrantly methylated cells, indicating that GOM occurs in the post-fertilization stage, especially after implantation.26, 27, 28

Epimutation of H19DMR is also a cause of SRS (OMIM #180860), which is characterized by opposite clinical phenotypes such as growth restriction.29 In ∼40% of SRS patients, methylated paternal H19DMR becomes hypomethylated (H19DMR-LOM), leading to increased H19 expression and decreased IGF2 expression.30 In contrast to BWS, essentially no mutations of H19DMR have been found in SRS patients with H19DMR-LOM. One SRS patient did exhibit a de novo mutation in H19DMR; however, as the mutation did not involve any putative protein-binding sites, it remains unknown if the mutation affected the methylation status of H19DMR.23 As a majority of cases with H19DMR-LOM show variable hypermethylation, LOM also occurs in the post-fertilization stage.29, 31

The CDKN1C/KCNQ1OT1 domain

The important genes in this domain are CDKN1C and KCNQ1OT1. CDKN1C encodes cyclin-dependent kinase inhibitor and shows preferential maternal expression. KCNQ1OT1 is a paternally-expressed, long non-coding RNA. The ICR of this domain is KvDMR1, located in intron 10 of the KCNQ1 gene, and it is methylated on the maternal but not the paternal allele (Figure 1). The methylation of KvDMR1 is established during oogenesis.17, 18 As KvDMR1 overlaps with the promoter of KCNQ1OT1, the paternal KCNQ1OT1 is expressed from unmethylated paternal KvDMR1 in the opposite direction of KCNQ1, and it functions to silence genes in the domain in cis.32 In mice, Kcnq1ot1 RNA interacts with G9a and the PRC2 complex, which mediates repressive histone modifications such as H3K9me3 and H3K27me3, and forms a repressive nuclear compartment that leads to gene silencing within the domain, including of Cdkn1c. However, this mechanism is specific to the placenta.33, 34 In mouse liver, Kcnq1ot1 RNA interacts with Dnmt1 to mediate maintenance of somatic DMRs, some of which overlap the Cdkn1c promoter, and silences genes within the domain.35 In addition, the identification of paternal allele-specific CTCF binding to KvDMR1 suggests that a repressive element within KvDMR1 likely regulated by CTCF acts to silence paternal Cdkn1c specifically and without promoter methylation in a subset of tissues (for example, kidney, liver and lung).36, 37 In humans, although KCNQ1OT1 coats the neighboring regions of chromatin-containing CDKN1C, the CDKN1C promoter does not show DMR, and H3K9me may not be involved in CDKN1C repression.38, 39 In two BWS families with significantly reduced expression of CDKN1C, maternal microdeletions for most parts of the KCNQ1 gene impact KvDMR1 and the following KCNQ1OT1 gene, but not CDKN1C, suggesting the presence of an enhancer element within the KCNQ1 locus for maternal expression of CDKN1C.40, 41 In addition, the DNA fragment containing KvDMR1 has been shown to have both silencer and insulator activities with CTCF binding.42 Therefore, researchers have proposed that CDKN1C is repressed on the paternal chromosome by KCNQ1OT1 RNA coating and by both a silencer and an insulator near KvDMR1, which is likely regulated by CTCF binding that prevents the CDKN1C promoter from accessing the enhancer downstream of KvDMR1.41

Loss of DNA methylation on the normally methylated maternal KvDMR1 (KvDMR1-LOM) accounts for ∼50% of BWS patients (Figure 1, Table 2). KvDMR1-LOM is accompanied by loss of H3K9me2, and this leads to expression of KCNQ1OT1 RNA, which in turn results in repression of CDKN1C expression on the maternal chromosome with the mechanism as proposed above.39, 41, 43, 44 In addition, only three families have been reported to have maternal transmission of the microdeletions containing KvDMR1, leading to reduced expression of CDKN1C.40, 41, 45 Such reduced expression induces the BWS phenotype.

Representative phenotypes of KvDMR1-LOM include omphalocele and hemihyperplasia (Table 2).12 As certain cases with isolated KvDMR1-LOM also display variable hypomethylation, patients are epigenetic mosaic, which indicates that LOM occurs in the post-fertilization stage.46, 47, 48, 49 Interestingly, monozygotic twins discordant for BWS are found predominantly for females. This could be in part explained by reduction of the amount of DNMT1 to maintain KvDMR1 methylation during the overlap in timing shared by X-inactivation and twinning.46

Paternal uniparental disomy

patUPD of 11p is found in ∼20% of patients (Figure 1, Table 2). All patients with patUPD are mosaic for patUPD cells and normal biparental cells, indicating occurrence of somatic recombination at the post-fertilization stage. Thus, UPD is always paternal isodisomy. Romanelli et al.50 analyzed nine patients with patUPD using SNP arrays, and found that the minimal patUPD size was ∼2.7 Mb from telomere to the centromeric side of KvDMR1 (Figure 1). As the minimal region includes both ICRs, H19DMR and KvDMR1, both H19DMR hypermethylation and KvDMR1 hypomethylation occur depending on the percentage of mosaicism and IGF2 overexpression; reduced expression of CDKN1C must be induced. Meanwhile, Romanelli et al.50 could not find hot-spots of mitotic recombination break points. One representative phenotypes of patUPD is hemihyperplasia, which can affect various regions of the body (Table 2).12

The largest patUPD size is the whole genome, denoted as genome-wide patUPD. Non-mosaic genome-wide patUPD results in hydatidiform mole formation. In contrast, individuals with mosaic genome-wide patUPD are born alive. To date, 11 patients with genome-wide patUPD have been reported.51, 52, 53, 54, 55, 56, 57, 58 Among these, half of the patients were diagnosed as BWS and only two displayed phenotypes associated with transient neonatal diabetes mellitus type 1 and upd(14)pat syndrome.51, 57 In addition, one patient with parthenogenic chimerism/mosaicism showed a SRS-like phenotype.59 These findings suggested an epi-dominant effect of aberrant methylation of 11p15 on clinical features. However, genome-wide patUPD patients with BWS phenotypes display atypical and varied phenotypes. This would be attributable to a paternal epigenotype for all ICRs and being homozygous for mutations of autosomal recessive genes. In addition, patients exhibit a significantly increased predisposition for tumor development. This also would be attributable to inactivation of tumor suppressor genes, or activation of oncogenes.

CDKN1C mutation

As mentioned before, CDKN1C is a gene responsible for the pathogenesis of BWS within the CDKN1C/KCNQ1OT1 domain, and it exhibits maternal preferential expression. This gene contains three exons divided by two introns encoding a 316 amino-acid protein, which is a strong inhibitor of several G1 cyclin/Cdk complexes and a negative regulator of cell proliferation.60, 61 The CDKN1C (p57KIP2) protein consists of three distinct domains: a cyclin-dependent kinase inhibitory domain, a proline and alanine repeat domain, and a QT domain (Figure 2). The cyclin-dependent kinase inhibitory domain contains a cyclin-binding region, a cyclin-dependent kinase-binding region and a 310 helix, which are both necessary and sufficient to bind and inhibit cyclin-dependent kinase activity.60, 61, 62 Proline and alanine repeats interact with the LIM domain kinase 1 and regulate actin dynamics.62, 63, 64 The QT domain contains a proliferating cell nuclear antigen (PCNA) binding domain, which can prevent DNA replication in vitro and S-phase entry in vivo, and a nuclear localization signal.60, 62, 65

Figure 2
figure 2

Mutations of CDKN1C in BWS and IMAGe syndrome.67, 68, 69 The mutations in BWS are loss-of-function mutations, which are either amino-acid substitution mutations localized to the cyclin-dependent kinase inhibitory domain or truncating mutations. The mutations in IMAGe syndrome that lead to growth restriction are missense mutations specific to the PCNA-binding domain, considered a gain-of-function mutation. Blue: amino-acid substitution mutations; red: truncating mutations.

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The mutations are found in ∼5% of sporadic cases, whereas dominant maternal transmission of germline mutations are found in 40% of familial BWS cases.11, 12 The mutations in sporadic cases should occur on the maternal allele because of maternal expression of CDKN1C. Approximately 30 mutations have been reported since the initial report by Hatada et al.66, 67, 68 These mutations are either missense mutations localized to the cyclin-dependent kinase inhibitory domain or nonsense mutations, both of which result in loss of function and lead to the BWS phenotype (Figure 2). Representative phenotypes of CDKN1C mutations include omphalocele and cleft palate (Table 2).12

Recently, missense mutations in the PCNA binding domain have reported in the undergrowth developmental disorder IMAGe syndrome (OMIM #614732), which is characterized by intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita, and genital anomalies (Figure 2).69 Only maternal transmission of the mutation results in IMAGe syndrome, consistent with imprinting inheritance. Targeted expression of patient-associated mutations in Drosophila caused restricted eye and wing growth, suggesting a gain-of-function mechanism. The gain of function might be due to abolishment of PCNA dependent CDKN1C monoubiquitination.69 It is intriguing that two opposite phenotypes, BWS and IMAGe syndrome, occur because of the mutations of the same CDKN1C gene. The biological role and molecular mechanism of the monoubiquitination should be elucidated to understand how the two disorders differ.

Chromosomal rearrangements

Chromosomal rearrangements involving 11p—including duplications, balanced translocations and inversions—occur in <2% of BWS patients (Figure 1, Table 2). Paternal duplications of 11p15 result in BWS due to overexpression of IGF2,70 whereas maternal duplications of 11p15 result in SRS.71 SRS and BWS phenotypes associated with 11p duplications in a single family have been reported.72 In this family, a SRS child was born from a mother with BWS phenotypes due to paternal duplication. Representative phenotypes of BWS due to duplication causes developmental delay (Table 2).12

So far at least 12 cases harboring translocations or inversions have been reported, with most break points of the translocations and inversions falling in the KCNQ1 locus.73, 74, 75, 76, 77 BWS develops when these are transmitted maternally. Three cases harboring inv(11)(p13;p15.5), inv(11)(p11.2;p15.5) and t(11;17)(p15.5;q21.3), respectively, have been seen to exhibit KvDMR1-LOM. However, a fibroblast with inv(11)(p15.5;q13) and a rhabdoid tumor line with t(11;22) have shown signs of reduced expression of CDKN1C with normal methylation at KvDMR1. These are consistent with the enhancer blocking insulator model mentioned before.75, 76, 77 However, the remaining cases showed neither KvDMR1-LOM nor reduced expression of CDKN1C. Therefore, the developmental mechanism for BWS harboring translocations and inversions is largely unknown.

Different risks for childhood tumors in each alteration type

Embryonal malignancies are the tumors most commonly associated with BWS—for example, Wilms’ tumor, hepatoblastoma, adrenocortical carcinoma, rhabdomyosarcoma and neuroblastoma—but other malignant or benign tumors are occasionally observed.6, 7 Although overall tumor risk is ∼7.5%, it is different for each causative alteration (Table 2). H19DMR-GOM and patUPD show the highest tumor risk, at >25%, especially for Wilms’ tumor and hepatoblastoma. KvDMR1-LOM has a rate of developing hepatoblastoma, rhabdomyosarcoma and gonadoblastoma other than Wilms’ tumors of ∼5%.10 The lowest risk is found in CDKN1C mutations with <5% of cases affected. Only neuroblastomas have been found in patients with CDKN1C mutations.78, 79 Wilms’ tumors are frequently seen in patients with H19DMR-GOM or patUPD, but never seen in patients with KvDMR1-LOM or CDKN1C mutations, suggesting a critical role of IGF2 overexpression in Wilms’ tumor development. In fact, IGF2 loss of imprinting is found in 60–70% of sporadic Wilms’ tumors without 11p LOH.80, 81 Furthermore, IGF2 loss of imprinting was also observed in ∼21% of sporadic hepatoblastomas without 11p LOH, and aberrant methylations at H19DMR, H19 promoter, IGF2-DMR0 or IGF2-DMR2 were observed in ∼55% of sporadic hepatoblastomas without 11p LOH, suggesting the importance of IGF2 overexpression for hepatoblastoma development as well (Rumbajan JM et al., submitted).82 In addition, although many kinds of adult tumors display reduced CDKN1C expression, of which certain cases show KvDMR1-LOM, the risk of embryonal tumorigenesis is low in BWS patients with KvDMR1-LOM or CDKN1C mutations, suggesting different contributions of CDKN1C to tumor development between adulthood and childhood.

ART and BWS

The worldwide usage of ART has increased. Several reports have raised concerns that the risk of imprinting disorders, such as BWS and Angelman syndrome, are increased in children conceived by ART, especially through in vitro fertilization and intracytoplasmic sperm injection, as the first reported associations in 2002 and 2003 between Angelman syndrome and BWS, respectively, with ART.83, 84, 85 The risk of BWS is estimated to be six to nine times higher in children conceived by ART than in children conceived naturally.86 The causative alteration for most of ART-related BWS is KvDMR1-LOM. The cause of Angelman syndrome is also LOM at SNRPN.

Animal studies have suggested that ovarian stimulation and culture medium for the embryo can affect DNA methylation and the expression of several imprinted genes.87, 88, 89, 90 In fact, ‘large offspring syndrome’ has been described as caused by LOM of the maternal Igf2r after sheep embryo culture.91 However, in humans, although ovarian stimulation may predispose to aberrant methylation at imprinted loci,92 it is still unclear whether the procedure of ART affects methylation at imprinted loci because ART populations are different from naturally conceived populations having low fertility rates, increased frequency of reproductive loss and advanced age.93 Indeed, male infertility is strongly associated with aberrant methylation at both maternal and paternal alleles.94, 95 It has been reported that there are no phenotypic differences between ART-related BWS and naturally conceived BWS.96 However, Lim et al.97 provided evidence that ART-related BWS had a significantly lower frequency of exomphalos and higher risk of tumor development than Wilms’ tumor. Larger size studies are needed to better understand the correlation between ART and BWS.

Multilocus hypomethylation disorders

Hypomethylations at several other imprinted loci have been reported to occur in BWS patients with KvDMR1-LOM.47, 48, 49, 97 As this phenomenon was also seen in patients with transient neonatal diabetes mellitus type 1 and SRS, a new entity of imprinting disorders such as MHD has been proposed.49, 98, 99, 100, 101 The literature indicates an overall frequency of multilocus hypomethylation in BWS patients with KvDMR1-LOM of 20% (49/244).49, 98, 99, 100, 101 IGF2R-DMR2, GNAS, NESPAS, PEG1 and PLAGL1 are frequently hypomethylated DMRs. In BWS patients, only maternally methylated DMRs displayed hypomethylation; however, several SRS patients with H19DMR-LOM showed hypomethylation at DLK1/GTL2 IG-DMR, another paternally methylated DMR, indicating involvement of both maternally and paternally methylated DMRs. In addition, a certain SRS showed hypomethylation at both H19DMR and KvDMR1.48, 100 As these hypomethylations were mosaic, they were presumed to be due to a post-fertilization event.

Lim et al.97 reported that ART-related BWS show multilocus hypomethylation more frequently than naturally conceived BWS; however, no such difference was observed by Rossignol et al.47 One study reported that BWS with multilocus hypomethylation displayed characteristics not usually associated with BWS, such as speech retardation, peri/postnatal apnea, feeding difficulties and hearing problems; additionally, nevus flammeus and hemihypertrophy were significantly lower in patients with multilocus hypomethylation.49 However, three other studies reported no difference in clinical features between MHDs and monolocus hypomethylation disorders.47, 48, 97 As the studies so far have analyzed only limited numbers of DMRs, further investigation of all known DMRs are needed.

The involvement of trans-acting factors in these MHD has been suggested. In fact, in one study, homozygous and compound heterozygous mutations of ZFP57, which encodes a KRAB zinc-finger protein and is required for the post-fertilization maintenance of maternal and paternal methylation imprinting at multiple loci, were found in transient neonatal diabetes mellitus type 1 patients with multilocus hypomethylation.102 However, no mutations were found in 27 BWS patients with KvDMR1-LOM probably without multilocus hypomethylation.103 KAP1, a protein associated with ZFP57, interacts with DNMT1 and binds to many ICRs in embryonic stem cells to maintain DNA and histone methylation.104, 105 Mice with maternal deletions of Trim28, a homolog of human KAP1, show aberrant DNA demethylation at a few ICRs.106 Mutation searches of KAP1 in MHD patients have not been reported to date.

Other candidates for trans-acting factors are NLRP2 and NLRP7, which are members of the Nod-like receptor protein (NLRP) family. Some NLRPs are components of the inflammasome, an assembly that is implicated in the sensing of, and inflammatory reaction to, extracellular pathogens and intracellular noxious compounds.107 Mutations of NLRP2 were identified in a familial case of BWS with KvDMR1-LOM and PEG1-LOM, suggesting a role of NLRP2 in the establishment or maintenance of ICRs.108 However, the mutation has not been corroborated by other studies yet. Mutations of NLRP7 and C6ORF221 account for familial biparental hydatidiform mole, which is a maternal effect recessive disorder resulting from failure of maternal imprints.109, 110 Mutation searches of NLRP7 were performed on the mother of a patient showing both transient neonatal diabetes mellitus type 1 and BWS features with multilocus hypomethylation, but they were unsuccessful.99 In addition, DNMT3L, which is required for establishing maternal imprints, was not mutated in two BWS patients with severe multilocus hypomethylation.49 Mutation searching of all candidate trans-acting factors should be performed over a large number of MHD patients to explore this matter further.

In addition, one circular chromosome conformation capture (4C) study revealed that maternal H19DMR interacts with the autosomal region, and imprinting domains were strongly overrepresented in the 4C library, suggesting the involvement of higher order chromatin interaction in the regulation of imprinting.111 The involvement of physical chromosome interactions in MHD should also be further elucidated.

Conclusions

Although H19DMR-GOM, KvDMR1-LOM, patUPD and CDKN1C mutations, and chromosomal rearrangements account for ∼80% of BWS phenotypes, several questions about these alterations still remain to be clarified. In addition, at least 20% of patients do not have these associated alterations, suggesting the existence of other, unknown epigenetic/genetic defects. Furthermore, other issues, such as the effect of ART on imprinting disorders and the mechanism of multilocus imprinting establishment/maintenance, should be clarified. Further investigations of all of these issues must be elucidated in order to understand the molecular basis of BWS and related imprinting disorders.