Epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is a model imprinting disorder resulting from mutations or epigenetic events involving imprinted genes at chromosome 11p15.5. Thus, germline mutations in CDKN1C, uniparental disomy (UPD), and loss of imprinting of IGF2 and other imprinted genes have been implicated. Many familial BWS cases have germline CDKN1C mutations. However, most BWS cases are sporadic and UPD or putative imprinting errors predominate in this group. We have identified previously a subgroup of sporadic cases with loss of imprinting (LOI) of IGF2 and epigenetic silencing of H19 proposed to be caused by a defect in a distal 11p15.5 imprinting control element (designated BWSIC1). However, many sporadic BWS patients show biallelic IGF2 expression in the presence of normal H19 methylation and expression patterns. This and other evidence suggested the existence of a further imprinting control element (BWSIC2) at 11p15.5. Recently, we showed that a subgroup of BWS patients have loss of methylation (LOM) at a differentially methylated region (KvDMR1) within the KCNQ1 gene centromeric to the IGF2 and H19 genes. We have now analysed a large series of sporadic cases to define the frequency and phenotypic correlates of epigenetic abnormalities in BWS. LOM at KvDMR1 was detected by Southern analysis or a novel PCR based method in 35 of 69 (51%) sporadic BWS without UPD. LOM at KvDMR1 was often, but not invariably associated with LOI of IGF2. KvDMR1 LOM was not detected in BWS patients with putative BWSIC1 defects and cases with KvDMR1 LOM (that is, putative BWSIC2 defects) invariably had a normal H19 methylation pattern. The incidence of exomphalos in putative BWSIC2 defect patients was not significantly different from that in patients with germline CDKN1C mutations (20/29 and 13/15 respectively), but was significantly greater than that in patients with putative BWSIC1 defects (0/5, p=0.007) and UPD (0/22, p<0.0001). These findings are consistent with the hypothesis that LOM of KvDMR1 (BWSIC2 defect) results in epigenetic silencing of CDKN1C and variable LOI of IGF2. BWS patients with embryonal tumours have UPD or a BWSIC1 defect but not LOM of KvDMR1. This study has further shown how (1) variations in phenotypic expression of BWS may be linked to specific molecular subgroups and (2) molecular analysis of BWS can provide insights into mechanisms of imprinting regulation.


Keywords: Beckwith-Wiedemann syndrome; epigenotype-phenotype correlations; imprinting     

Alternative mechanisms associated with silencing of CDKN1C in Beckwith-Wiedemann syndrome

Background: Mutations in the imprinted gene CDKN1C account for approximately 10% of Beckwith–Wiedemann syndrome (BWS) cases. Fibroblasts from BWS patients with loss of methylation (LOM) at the imprinting control region (ICR) KvDMR1 have reduced CDKN1C expression. Another group of BWS patients with downregulated CDKN1C expression but with normal methylation at KvDMR1 has been identified. Objective: To investigate the mechanism of CDKN1C silencing in BWS in these two classes of patients. Methods: The CDKN1C promoter region was analysed for changes in DNA methylation using bisulphite sequencing, and for alterations in chromatin structure using the chromatin immunoprecipitation (ChIP) assay. Results: There was only spurious CpG methylation of the CDKN1C promoter in fibroblast DNA from both normal individuals and patients with BWS, irrespective of the methylation status of KvDMR1. There was no detectable change in chromatin structure at the CDKN1C promoter in patients with LOM at KvDMR1. BWS patients with downregulated CDKN1C and normal methylation at KvDMR1 had depletion of dimethylated H3-K4 and enrichment of dimethylated H3-K9 and HP1γ at the CDKN1C promoter, suggesting that in these cases gene silencing is associated with repressive chromatin changes. Conclusions: CDKN1C may be downregulated by multiple mechanisms including some that do not involve promoter methylation. In BWS patients with normal methylation at KvDMR1 and reduced expression of CDKN1C, repressive chromatin may play a role, but the absence of methylation and repressive chromatin structure at the CDKN1C promoter in BWS patients with LOM at KvDMR1 argues for a direct role of this epimutation in silencing CDKN1C.     

Silencing of CDKN1C (p57KIP2) is associated with hypomethylation at KvDMR1 in Beckwith-Wiedemann syndrome

Context: Beckwith–Wiedemann syndrome (BWS) arises by several genetic and epigenetic mechanisms affecting the balance of imprinted gene expression in chromosome 11p15.5. The most frequent alteration associated with BWS is the absence of methylation at the maternal allele of KvDMR1, an intronic CpG island within the KCNQ1 gene. Targeted deletion of KvDMR1 suggests that this locus is an imprinting control region (ICR) that regulates multiple genes in 11p15.5. Cell culture based enhancer blocking assays indicate that KvDMR1 may function as a methylation modulated chromatin insulator and/or silencer.

Objective: To determine the potential consequence of loss of methylation (LOM) at KvDMR1 in the development of BWS.

Methods: The steady state levels of CDKN1C gene expression in fibroblast cells from normal individuals, and from persons with BWS who have LOM at KvDMR1, was determined by both real time quantitative polymerase chain reaction (qPCR) and ribonuclease protection assay (RPA). Methylation of the CDKN1C promoter region was assessed by Southern hybridisation using a methylation sensitive restriction endonuclease.

Results: Both qPCR and RPA clearly demonstrated a marked decrease (86–93%) in the expression level of the CDKN1C gene in cells derived from patients with BWS, who had LOM at KvDMR1. Southern analysis indicated that downregulation of CDKN1C in these patients was not associated with hypermethylation at the presumptive CDKN1C promoter.

Conclusions: An epimutation at KvDMR1, the absence of maternal methylation, causes the aberrant silencing of CDKN1C, some 180 kb away on the maternal chromosome. Similar to mutations at this locus, this silencing may give rise to BWS.

     

Analysis of CDKN1C in Beckwith Wiedemann syndrome

In this study we have examined 32 patients with Beckwith Wiedemann Syndrome (BWS) for mutations affecting the CDKN1C gene, including seven cases of familial BWS. Mutations were not detected in the coding region of the CDKN1C gene in any individual with BWS. However in two patients, two G/A base substitutions at adjacent positions in the 5'UTR were detected. These substitutions were also found in normal controls. Expression of CDKN1C in somatic tissues was examined in 18 of the 32 cases using semi-quantitative RT-PCR. CDKN1C expression was significantly reduced in the peripheral blood of three cases compared with controls. These results suggest that, although coding region mutations in the CDKN1C gene are rare in BWS, mutations disrupting CDKN1C expression may be found. Three of five informative patients exhibited biallelic CDKN1C expression in lymphocytes, cord blood, and kidney tissue, respectively. Biallelic expression was not associated with overall CDKN1C levels significantly different to those in controls. Patients who expressed CDKN1C biallelically, or who were low CDKN1C expressors, maintained monoallelic methylation in the Differentially Methylated Region 2 (DMR2) of the IGF2 locus. One patient expressing CDKN1C biallelically, maintained imprinted gene expression at the IGF2 locus. These results suggest that biallelic CDKN1C expression does not significantly perturb the overall levels of CDKN1C expression in somatic tissue. They also confirm other studies showing that the mechanisms associated with regulating CDKN1C expression and imprinting are separate from those regulating IGF2 imprinting.     

Japanese and North American/European patients with Beckwith-Wiedemann syndrome have different frequencies of some epigenetic and genetic alterations

Beckwith-Wiedemann syndrome (BWS) is an imprinting-related human disease. The frequencies of causative alterations such as loss of methylation (LOM) of KvDMR1, hypermethylation of H19-DMR, paternal uniparental disomy, CDKN1C gene mutation, and chromosome abnormality have been described for North American and European patients, but the corresponding frequencies in Japanese patients have not been measured to date. Analysis of 47 Japanese cases of BWS revealed a significantly lower frequency of H19-DMR hypermethylation and a higher frequency of chromosome abnormality than in North American and European patients. These results suggest that susceptibility to epigenetic and genetic alterations differs between the two groups.     

Analysis of germline CDKN1C (p57KIP2) mutations in familial and sporadic Beckwith-Wiedemann syndrome (BWS) provides a novel genotype-phenotype correlation.

Beckwith-Wiedemann syndrome (BWS) is a human imprinting disorder with a variable phenotype. The major features are anterior abdominal wall defects including exomphalos (omphalocele), pre- and postnatal overgrowth, and macroglossia. Additional less frequent complications include specific developmental defects and a predisposition to embryonal tumours. BWS is genetically heterogeneous and epigenetic changes in the IGF2/H19 genes resulting in overexpression of IGF2 have been implicated in many cases. Recently germline mutations in the cyclin dependent kinase inhibitor gene CDKN1C (p57KIP2) have been reported in a variable minority of BWS patients. We have investigated a large series of familial and sporadic BWS patients for evidence of CDKN1C mutations by direct gene sequencing. A total of 70 patients with classical BWS were investigated; 54 were sporadic with no evidence of UPD and 16 were familial from seven kindreds. Novel germline CDKN1C mutations were identified in five probands, 3/7 (43%) familial cases and 2/54 (4%) sporadic cases. There was no association between germline CDKN1C mutations and IGF2 or H19 epigenotype abnormalities. The clinical phenotype of 13 BWS patients with germline CDKN1C mutations was compared to that of BWS patients with other defined types of molecular pathology. This showed a significantly higher frequency of exomphalos in the CDKN1C mutation cases (11/13) than in patients with an imprinting centre defect (associated with biallelic IGF2 expression and H19 silencing) (0/5, p<0.005) or patients with uniparental disomy (0/9, p<0.005). However, there was no association between germline CDKN1C mutations and risk of embryonal tumours. No CDKN1C mutations were identified in six non-BWS patients with overgrowth and Wilms tumour. These findings (1) show that germline CDKN1C mutations are a frequent cause of familial but not sporadic BWS, (2) suggest that CDKN1C mutations probably cause BWS independently of changes in IGF2/H19 imprinting, (3) provide evidence that aspects of the BWS phenotype may be correlated with the involvement of specific imprinted genes, and (4) link genotype-phenotype relationships in BWS and the results of murine experimental models of BWS.     

Molecular subtypes and phenotypic expression of Beckwith-Wiedemann syndrome

Beckwith-Wiedemann Syndrome (BWS) results from mutations or epigenetic events involving imprinted genes at 11p15.5. Most BWS cases are sporadic and uniparental disomy (UPD) or putative imprinting errors predominate in this group. Sporadic cases with putative imprinting defects may be subdivided into (a) those with loss of imprinting (LOI) of IGF2 and H19 hypermethylation and silencing due to a defect in a distal 11p15.5 imprinting control element (IC1) and (b) those with loss of methylation at KvDMR1, LOI of KCNQ1OT1 (LIT1) and variable LOI of IGF2 in whom there is a defect at a more proximal imprinting control element (IC2). We investigated genotype/epigenotype-phenotype correlations in 200 cases with a confirmed molecular genetic diagnosis of BWS (16 with CDKN1C mutations, 116 with imprinting centre 2 defects, 14 with imprinting centre 1 defects and 54 with UPD). Hemihypertrophy was strongly associated with UPD (P<0.0001) and exomphalos was associated with an IC2 defect or CDKN1C mutation but not UPD or IC1 defect (P<0.0001). When comparing birth weight centile, IC1 defect cases were significantly heavier than the patients with CDKN1C mutations or IC2 defect (P=0.018). The risk of neoplasia was significantly higher in UPD and IC1 defect cases than in IC2 defect and CDKN1C mutation cases. Kaplan-Meier analysis revealed a risk of neoplasia for all patients of 9% at age 5 years, but 24% in the UPD subgroup. The risk of Wilms' tumour in the IC2 defect subgroup appears to be minimal and intensive screening for Wilms' tumour appears not to be indicated. In UPD patients, UPD extending to WT1 was associated with renal neoplasia (P=0.054). These findings demonstrate that BWS represents a spectrum of disorders. Identification of the molecular subtype allows more accurate prognostic predictions and enhances the management and surveillance of BWS children such that screening for Wilms' tumour and hepatoblastoma can be focused on those at highest risk.     

No evidence for pathogenic variants or maternal effect of ZFP57 as the cause of Beckwith-Wiedemann Syndrome

Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome, which, in 50-60% of sporadic cases, is caused by hypomethylation of KCNQ1OT1 differentially methylated region (DMR) at chromosome 11p15.5. The underlying defect of this hypomethylation is largely unknown. Recently, recessive mutations of the ZFP57 gene were reported in patients with transient neonatal diabetes mellitus type 1, showing hypomethylation at multiple imprinted loci, including KCNQ1OT1 DMR in some. The aim of our study was to determine whether ZFP57 alterations were a genetic cause of the hypomethylation at KCNQ1OT1 DMR in patients with BWS. We sequenced ZFP57 in 27 BWS probands and in 23 available mothers to test for a maternal effect. We identified three novel, presumably benign sequence variants in ZFP57; thus, we found no evidence for ZFP57 alterations as a major cause in sporadic BWS cases.     

DNA methylation studies on imprinted loci in a male monozygotic twin pair discordant for Beckwith–Wiedemann syndrome

Tierling S, Souren NY, Reither S, Zang KD, Meng‐Hentschel J, Leitner D, Oehl‐Jaschkowitz B, Walter J. DNA methylation studies on imprinted loci in a male monozygotic twin pair discordant for Beckwith–Wiedemann syndrome. Beckwith–Wiedemann syndrome (BWS) is one of the most prevalent congenital disorders predominantly caused by epigenetic alterations. Here we present an extensive case study of a monozygotic monochorionic male twin pair discordant for BWS. Our analysis allows to correlate BWS symptoms, like a protruding tongue, indented ears and transient neonatal hypoglycaemia, to an abnormal methylation at the KvDMR1. DNAs extracted from peripheral blood, skin fibroblasts, saliva and buccal swab of both twins, their sister and parents were analysed at 11 differentially methylated regions (DMRs) including all four relevant DMRs of the BWS region. The KvDMR1 was exclusively found to be hypomethylated in all cell types of the affected BWS twin, while the unaffected twin and the relatives showed normal methylation in fibroblasts, buccal swab and saliva DNA. Interestingly, the twins share a common blood‐specific hypomethylation phenotype most probably caused by a feto‐fetal transfusion between both twins. Because microsatellite analysis furthermore revealed a normal biparental karyotype for chromosome 11, our results point to an exclusive correlation of the observed BWS symptoms to locally restricted epimutations at the KvDMR1 of the maternal chromosome.     

Allelic methylation of H19 and IGF2 in the Beckwith -- Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) Is a congenital overgrowthsyndrome with associated embryonal tumours. Most BWS cases aresporadic but familial cases occur In 15% of patients and inthese there is linkage to chromosome 11p15. In addition, a smallnumber of patients have cytogenetic abnormalities involvingchromosome 11 p15. Approximately 20% of sporadic BWS patientshave uniparental paternal dlsomy (UPD) of chromosome 11 p15.This finding together with the observation that penetrance infamilial cases depends on parental transmission, suggests thatthe gene(s) for BWS are imprinted. The recent demonstrationof blallellc expression of the otherwise maternally imprintedIGF2 gene In some BWS patients implicates excess IGF2 expressionin the disease. Here we have analysed the allele-specific methylationpatterns In the IGF2 gene and in the neighbouring and reciprocallyImprinted H19 gene in a group of 42 BWS patients, 10 of whichwere mosaic UPD cases. We found that allelic methylation ofboth genes was normal in all non-UPD cases, with the paternalallele being methylated, and was Increased In UPD cases in proportionwith the disomlc lineage. These findings suggest that sporadicBWS is not associated with a general alteration of methylationimprinting of the IGF2 and H19 genes. The methylation assayused in this study thus also offers a simple and reliable diagnostictest of UPD for 11p15.5. An unexpected finding was a distortionof the frequency of AvaU alleles at the IGF2 locus exclusivelyin UPD BWS cases (P < 0.001). This further Implicates theIGF2 gene in aspects of the BWS phenotype.     

Rapid detection of methylation change at H19 in human imprinting disorders using methylation-sensitive high-resolution melting

Beckwith Wiedemann syndrome (BWS) and Russell Silver syndrome (RS) are growth disorders with opposing epimutations affecting the H19/IGF2 imprinting center at 11p15.5. Overgrowth and tumor risk in BWS is caused by aberrant expression of the paternally expressed, imprinted IGF2 gene, occurring as a consequence of mosaic hypermethylation within the imprinting center, or to mosaic paternal uniparental disomy (UPD). RS is characterized by severe intrauterine growth retardation (IUGR). A subset of RS cases were recently shown to have mosaic hypomethylation within the H19/IGF2 imprinting center, predicted to silence paternally expressed IGF2 in early development. Molecular diagnosis for BWS and RS involves methylation analysis of the H19 locus, enabling discrimination of allelic methylation patterns. In this study, methylation-sensitive high-resolution melting analysis (MS-HRM) was used to analyze methylation within the intergenic region of the H19 locus. A total of 36 samples comprising normal control (11), BWS (19), and RS (six) DNA were analyzed in a blinded study and scored as hypermethylated, normal, or hypomethylated. Results were compared with those derived by methylation-sensitive Southern blotting using the restriction enzymes Rsa I and Hpa II. A total of 100% concordance was obtained for the Southern blotting and MS-HRM scores. A total of three samples with paternal duplication affecting the H19/IGF2 region were scored as equivocal by both methods; however, 33 out of 36 (92%) the samples were unambiguously scored as being hypermethylated, hypomethylated, or normally methylated using MS-HRM. We conclude that MS-HRM is a rapid, cost-effective, and sensitive method for screening mosaic methylation changes at the H19 locus in BWS and RS.     

Comprehensive and quantitative multilocus methylation analysis reveals the susceptibility of specific imprinted differentially methylated regions to aberrant methylation in Beckwith–Wiedemann syndrome with epimutations

PurposeExpression of imprinted genes is regulated by DNA methylation of differentially methylated regions (DMRs). Beckwith–Wiedemann syndrome is an imprinting disorder caused by epimutations of DMRs at 11p15.5. To date, multiple methylation defects have been reported in Beckwith–Wiedemann syndrome patients with epimutations; however, limited numbers of DMRs have been analyzed. The susceptibility of DMRs to aberrant methylation, alteration of gene expression due to aberrant methylation, and causative factors for multiple methylation defects remain undetermined.MethodsComprehensive methylation analysis with two quantitative methods, matrix-assisted laser desorption/ionization mass spectrometry and bisulfite pyrosequencing, was conducted across 29 DMRs in 54 Beckwith–Wiedemann syndrome patients with epimutations. Allelic expressions of three genes with aberrant methylation were analyzed. All DMRs with aberrant methylation were sequenced.ResultsThirty-four percent of KvDMR1–loss of methylation patients and 30% of H19DMR–gain of methylation patients showed multiple methylation defects. Maternally methylated DMRs were susceptible to aberrant hypomethylation in KvDMR1–loss of methylation patients. Biallelic expression of the genes was associated with aberrant methylation. Cis-acting pathological variations were not found in any aberrantly methylated DMR.ConclusionMaternally methylated DMRs may be vulnerable to DNA demethylation during the preimplantation stage, when hypomethylation of KvDMR1 occurs, and aberrant methylation of DMRs affects imprinted gene expression. Cis-acting variations of the DMRs are not involved in the multiple methylation defects.Genet Med16 12, 903–912.     

Beckwith-Wiedemann-like macroglossia and 18q23 haploinsufficiency

Beckwith-Wiedemann syndrome (BWS) is an overgrowth condition with tumor proclivity linked to a genetic imbalance of a complex imprinted region in 11p15.5. A female child with features fitting in with the BWS diagnostic framework and an apparent loss of imprinting (LOI) of the IGF2 gene in 11p15.5 was also reported to have a de novo chromosome 18q segmental deletion (Patient 1), thus pointing at the location of a possible trans-activating regulator element for maintenance of IGF2 imprinting and providing one of the few examples of locus heterogeneity of BWS. A second child with de novo 18q23 deletion and features of macroglossia, naevus flammeus, bilateral inguinal hernia and transient neonatal hypoglycemia, thus also fitting in with the BWS diagnostic framework, is here fully reported (Patient 2). In this child, an analysis of the BWS1 locus precluded any paternal isodisomy and showed a normal imprinting pattern (mono-allelic expression of IGF2 and normal H19 and CDKN1OT1/LIT1 methylation index). In Patients 1 and 2, deletions were shown to overlap, defining a minimal region of haplo-insufficiency of 3.8-5.6 Mb in 18q23. We conclude that this region provides a candidate location for an original macroglossia condition with strong overlap with BWS, but without obvious upstream functional relationship with the BWS1 locus in 11p15.5. Because this minimal region of haplo-insufficiency falls into a common region of deletion in 18q- syndrome, we inferred that this macroglossia condition would follow a recessive pattern of inheritance.     

Positive associations between single nucleotide polymorphisms in the IGF2 gene region and body mass index in adult males.

We previously demonstrated an association between the insulin-like growth factor 2 (IGF2) gene 3'-untranslated region (3'-UTR) ApaI polymorphism and body mass index (BMI) in over 2500 middle-aged Caucasoid males. In the same cohort, we have now tested association with 11 more markers, including seven novel single nucleotide polymorphisms (SNPs), spanning >30 kb across the IGF2 gene. Three SNPs showed significant positive associations with BMI: 6815 A/T in the IGF2 P1 promoter (P = 0.00012, n = 2394) and the newly identified SNPs 1156 C/T in intron 2 (P = 0.017, n = 1567) and 1926 C/G in the 3'-UTR (P = 0.0062, n = 1872). There was strong pairwise linkage disequilibrium (LD) between the ApaI and 1926 C/G sites, whereas LD between ApaI and 6815 A/T, and between ApaI and 1156 T/C, was minimal. Univariately 6815 A/T, 1156 T/C and ApaI explained 1.03, 1.02 and 0.67% of the variation in BMI. Multi-way analysis of variance (ANOVA) models showed that 6815 A/T and 1156 T/C explained a further 0.4 and 0.8% of the variation beyond that accounted for by ApaI and the association of 1926 C/G with BMI disappeared after adjustment. The 6815 A/T, 1156 T/C and ApaI markers in effect constitute independent affirmations of our original hypothesized candidate gene region. In a stepwise multi-way ANOVA model, all three terms were significantly independently associated with BMI. The total proportion of BMI variance explained by this model was 2.25%, strongly suggesting that IGF2 genetic variation is a significant determinant of body weight in middle-aged males.     

The epigenetic imprinting defect of patients with Beckwith-Wiedemann syndrome born after assisted reproductive technology is not restricted to the 11p15 region

Background

Genomic imprinting refers to an epigenetic marking resulting in monoallelic gene expression and has a critical role in fetal development. Various imprinting diseases have recently been reported in humans and animals born after the use of assisted reproductive technology (ART). All the epimutations implicated involve a loss of methylation of the maternal allele (demethylation of KvDMR1/KCNQ1OT1 in Beckwith–Wiedemann syndrome (BWS), demethylation of SNRPN in Angelman syndrome and demethylation of DMR2/IGF2R in large offspring syndrome), suggesting that ART impairs the acquisition or maintenance of methylation marks on maternal imprinted genes. However, it is unknown whether this epigenetic imprinting error is random or restricted to a specific imprinted domain.

Aim

To analyse the methylation status of various imprinted genes (IGF2R gene at 6q26, PEG1/MEST at 7q32, KCNQ1OT1 and H19 at 11p15.5, and SNRPN at 15q11–13) in 40 patients with BWS showing a loss of methylation at KCNQ1OT1 (11 patients with BWS born after the use of ART and 29 patients with BWS conceived naturally).

Results

3 of the 11 (27%) patients conceived using ART and 7 of the 29 (24%) patients conceived normally displayed an abnormal methylation at a locus other than KCNQ1OT1.

Conclusions

Some patients with BWS show abnormal methylation at loci other than the 11p15 region, and the involvement of other loci is not restricted to patients with BWS born after ART was used. Moreover, the mosaic distribution of epimutations suggests that imprinting is lost after fertilisation owing to a failure to maintain methylation marks during pre‐implantation development.Genomic imprinting refers to an epigenetic marking of certain genes, resulting in monoallelic expression in a parent‐of‐origin‐dependent manner. Target gene expression on imprinted regions involves changes in chromatin structure and specific patterns of DNA methylation and post‐translational histone modifications such as acetylation and methylation.¹ Imprinting control elements are characterised by differentially methylated regions (DMR), in which the imprinted allele is methylated and the other parental allele is unmethylated. Imprinting is established during the development of germ cells and must be maintained at a critical stage of pre‐implantation development when the rest of the genome is subjected to a wave of demethylation.¹Imprinted genes have a crucial role in mammalian development particularly in fetal growth. Aberrant imprinting results in numerous human genetic disorders, including behavioural disorders and cancer. Syndromes involving epigenetic changes have recently been reported in animals and humans conceived by assisted reproductive technology (ART). These syndromes include large offspring syndrome in ruminants,² and the Beckwith–Wiedemann syndrome (BWS; MIM 130650)^3,4,5,6 and Angelman syndrome (MIM 105830)^7,8 in humans. Many genetic and epigenetic mechanisms are involved in Angelman syndrome and BWS, but, remarkably, in all imprinting disorders observed after the use of ART, the epimutation involves a loss of methylation at maternally imprinted methylated imprinting control elements (IGF2R DMR2 (MIM 147280) in large offspring syndrome, KvDMR1 of the KCNQ1OT1 gene (MIM 604115) in patients with BWS and SNURF‐SNRPN exon 1/promoter (MIM 182279) in those with Angelman syndrome). The demethylation of these imprinting control elements is responsible for a maternal to paternal switch, with activation of non‐coding RNA on the maternal allele.The precise mechanism underlying the association of imprinted disorders and ART is unknown. The gamete and embryo manipulations used in ART may interfere with genomic imprinting by altering the acquisition or maintenance of imprints during germ‐cell maturation or early embryogenesis. In mice, maternal methylation marks are acquired sequentially by the various maternal imprinted loci during oocyte growth.⁹ Alternatively, ART may alter the maintenance of methylation imprints in pre‐implantation embryos. No specific procedure has yet been implicated in the epigenetic risk of babies conceived by ART. Indeed, the ART used to conceive patients with Angelman syndrome and BWS involved various procedures, including classical in vitro fertilisation, intracytoplasmic sperm injection (ICSI), embryo cryopreservation and early or late embryo transfer.^3,4,5,6,7,8 However, two recent studies^10,11 have suggested that ovarian stimulation may itself increase the risk of Angelman syndrome and BWS. Moreover, the epigenetic change in patients with BWS conceived by ART is the same as that found in monozygotic twins with BWS. The prevalence of monozygotic twinning is particularly high in patients with BWS.¹² Twins are discordant and the affected twin always shows demethylation of KCNQ1OT1,^12,13 suggesting that the KCNQ1OT1 locus is vulnerable to demethylation at a critical stage of pre‐implantation development.No attempt has yet been made to determine whether the epigenetic imprinting error after the use of ART is restricted to a specific imprinted domain or is randomly distributed. In this study, we analysed the methylation status of other imprinted loci in patients with BWS conceived by ART. We showed that some patients with BWS born after using ART and some patients with BWS conceived naturally displayed abnormal methylation at loci other than the 11p15 centromeric domain.     

CDKN1C expression in Beckwith-Wiedemann syndrome patients with allele imbalance

In this study, we have examined CDKN1C expression in BWS patients with allele imbalance (AI) affecting the 11p15 region. Two of two informative patients with AI, attributable to mosaic paternal isodisomy, exhibited reduced levels of CDKN1C expression in the liver and kidney, respectively, relative to expression levels in the equivalent tissues in normal controls. Although overall expression was reduced, some expression from the paternally derived CDKN1C allele was evident, consistent with incomplete paternal imprinting of the gene. One patient showed evidence of maternal allele silencing in addition to AI. These findings show for the first time that CDKN1C expression is reduced in BWS patients with AI and suggest that CDKN1C haploinsufficiency contributes to the BWS phenotype in patients with mosaic paternal isodisomies of chromosome 11.