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Human Molecular Genetics Advance Access originally published online on February 1, 2008
Human Molecular Genetics 2008 17(10):1427-1435; doi:10.1093/hmg/ddn031
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Different mechanisms cause imprinting defects at the IGF2/H19 locus in Beckwith–Wiedemann syndrome and Wilms' tumour

Flavia Cerrato1,2, Angela Sparago1,2, Gaetano Verde1,2, Agostina De Crescenzo1,2, Valentina Citro3, Maria Vittoria Cubellis3, Maria Michela Rinaldi5, Luigi Boccuto6, Giovanni Neri6, Cinzia Magnani7, Paolo D'Angelo8, Paola Collini9, Daniela Perotti10, Gianfranco Sebastio4, Eamonn R. Maher11 and Andrea Riccio1,*

1 Dipartimento di Scienze Ambientali, Seconda Università di Napoli, via Vivaldi 43, 81100 Caserta, Italy 2 Institute of Genetics and Biophysics ‘A. Buzzati Traverso’, CNR, Naples, Italy 3 Dipartimento di Biologia Strutturale e Funzionale 4 Dipartimento di Pediatria, Università di Napoli ‘Federico II’, Naples, Italy 5 U.O.C. Genetica Medica, A.O.R.N.A. Cardarelli, Naples, Italy 6 Institute of Medical Genetics, Università Cattolica del Sacro Cuore, Rome, Italy 7 Dipartimento Materno-Infantile, Azienda Ospedale-Università, Parma, Italy 8 Unit of Paediatric Hematology and Oncology, ‘G. Di Cristina’ Children Hospital, Palermo, Italy 9 Department of Anatomic Pathology 10 Department of Experimental Oncology and Laboratories, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 11 Medical and Molecular Genetics Section, University of Birmingham, The Medical School, Birmingham, UK

* To whom correspondence should be addressed. Tel: +39 0823274599; Fax: +39 082274605; Email: andrea.riccio{at}unina2.it

Received November 19, 2007; Accepted January 29, 2008


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 ELECTRONIC-DATABASE INFORMATION
 FUNDING
 REFERENCES
 
The parent of origin-dependent expression of the IGF2 and H19 genes is controlled by the imprinting centre 1 (IC1) consisting in a methylation-sensitive chromatin insulator. Deletions removing part of IC1 have been found in patients affected by the overgrowth- and tumour-associated Beckwith–Wiedemann syndrome (BWS). These mutations result in the hypermethylation of the remaining IC1 region, loss of IGF2/H19 imprinting and fully penetrant BWS phenotype when maternally transmitted. We now report that 12 additional cases with IC1 hypermethylation have a similar clinical phenotype but showed neither a detectable deletion nor other mutation in the local vicinity. Likewise, no IC1 deletion was detected in 40 sporadic non-syndromic Wilms' tumours. A detailed analysis of the BWS patients showed that the hypermethylation variably affected the IC1 region and was generally mosaic. We observed that all these cases were sporadic and in at least two families affected and unaffected members shared the same maternal IC1 allele but not the abnormal maternal chromosome epigenotype. Furthermore, the chromosome with the imprinting defect derived from either the maternal grandfather or maternal grandmother. Overall, these results indicate that methylation-imprinting defects at the IGF2–H19 locus can result from inherited mutations of the IC and have high recurrence risk or arise independently from the sequence context and generally not transmitted to the progeny. Despite these differences, the epigenetic abnormalities are usually present in the patients in the mosaic form and probably acquired by post-zygotic de novo methylation. Distinguishing between these two groups of cases is important for genetic counselling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 ELECTRONIC-DATABASE INFORMATION
 FUNDING
 REFERENCES
 
There is increasing evidence that aberrant chromatin states leading to aberrant gene expression patterns (epimutations) have important roles in human disease (1,2). However, many characteristics of these lesions are still undefined. For instance, although it is generally accepted that epigenetic marks are cleared between generations, there are a number of cases in which this seems not to be the case. Indeed, the causes, heritability and relationship with phenotype of some epimutations have recently been the matter of debate (3–7). The human disorders caused by defects of genomic imprinting provide a paradigm for studying these issues.

Genomic imprinting is a mechanism causing the expression of a minority of genes to be monoallelic and dependent on their gametic origin (8,9). Imprinted genes are often organized in clusters and their gamete of origin-specific expression is controlled by cis-acting elements termed imprinting centres (ICs). These sequences are marked by different epigenetic modifications (of which DNA methylation is an essential one) on their maternal and paternal alleles. These molecular imprints are established during gametogenesis, maintained in the zygote and throughout embryo development and replaced by the new ones in the new generation of germ cells. In the somatic cells, the ICs direct by different mechanisms the parent of origin-specific expression of the imprinted genes. A 1 Mb cluster of imprinted genes lies on chromosome 11p15.5. This cluster is functionally divided into two domains that are autonomously controlled by distinct ICs [IC1 and IC2 (10,11)]. Defective expression of the 11p15.5 imprinted genes results in the Beckwith–Wiedemann syndrome (BWS, MIM 130650 [OMIM] ).

BWS is a developmental disorder characterized by variable clinical features, including overgrowth, macroglossia, abdominal wall defects and increased incidence of embryonal tumours (12). The majority of the BWS cases are sporadic. The rare familial cases show predominantly autosomal dominant inheritance and preferential expression following maternal transmission. Heterogeneous molecular defects are found in BWS (12). Only 5% of the cases (40% of the familial ones) have typical single-gene defects, consisting in loss-of-function mutations of CDKN1C. Approximately 20% of the cases have uniparental paternal disomy (UPD) of 11p15.5 loci, indicating that BWS is caused by excess of imprinted genes expressed from the paternal chromosome and/or defect of imprinted genes expressed from the maternal chromosome. The majority of the other cases show DNA methylation defects at either IC1 or IC2. These are accompanied by deregulation (either biallelic activation or biallelic silencing) of Domain 1 or Domain 2 imprinted genes. Microdeletions of the ICs that account for the regulatory defects have been found in some of these patients (13–17). Although many phenotypic characteristics are in common between different molecular subtypes of BWS, significant differences have been found for some of the clinical features of BWS, such as tumour risk, severity of anterior abdominal wall defect, birth weight, hemihypertrophy and ear signs (18–20).

Two genes, insulin-like growth factor 2 (IGF2) and H19, are located in Domain 1 of the 11p15.5 imprinted gene cluster. IGF2 is a paternally expressed fetal growth factor. H19 is a maternally expressed non-coding RNA with possible tumour-suppressor functions (21). The reciprocal imprinting of IGF2 and H19 is controlled by IC1 in the majority of tissues. The function of this control element has been extensively studied in the mouse. IC1 [also known as H19 differentially methylated region (DMR)] is a methylation-sensitive chromatin insulator that is located between Igf2 and H19 (22,23). The maternal and paternal IC1 alleles acquire differential methylation during gametogenesis. In the somatic cells, the non-methylated maternal allele interacts with the zinc-finger protein CTCF. This binding is required for preventing de novo methylation of the region, insulating the maternal Igf2 promoter from downstream enhancers and activating the maternal H19 gene. On the paternal chromosome, instead, DNA methylation prevents CTCF binding at IC1 and allows Igf2 activation by the enhancers while the paternal H19 promoter becomes hypermethylated and silenced. Recent evidence indicates that CTCF binding at IC1 mediates higher-order chromatin conformations in a parent of origin-specific manner (24–26). This may partition the paternal and maternal Igf2 alleles into active and inactive chromatin domains, respectively. Five to ten percent of the patients with BWS have methylation of IC1 on both parental chromosomes. This results in biallelic activation of IGF2 and biallelic silencing of H19 in all tissues (27). Patients with this type of defect have higher risk of developing cancer and particularly Wilms' tumour (WT) than patients belonging to other molecular subgroups (18). Similar to BWS, patients with non-syndromic WT also have IC1 hypermethylation with IGF2 activation and H19 silencing, but this is restricted to cancer tissues in these cases (28).

Deletions of the IC1 region of the order of 1.4–1.8 kb have been found in BWS patients with IC1 hypermethylation (13,16,17). These cases are part of dominant inheritance pedigrees with maternal transmission. The deletions remove one to two target sequences for CTCF (CTSs) resulting in hypermethylation of the residual CTSs and co-segregate with the BWS phenotype. We found no deletion in other phenotypically similar BWS cases. In this paper, we investigate the cause of defective imprinting of IGF2–H19 in 12 individuals with IC1 hypermethylation and no associated deletion. The results obtained suggest that these cases result from sporadic epimutations arising independently from the sequence context, probably at post-zygotic stages and have low recurrence risk. Similarly, sporadic methylation defects without accompanying IC1 deletion were found in a large series of non-syndromic WTs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 ELECTRONIC-DATABASE INFORMATION
 FUNDING
 REFERENCES
 
Search for IC1 deletions
We identified 21 individuals with the clinical features of BWS and hypermethylation of the IC1 and H19 promoter regions as determined by Southern blotting (data not shown). Nine of these patients carried IC1 deletions and were part of five dominant pedigrees with maternal transmission (13,17). No deletion in the IC1 region was identified in the remaining 12 individuals by Southern blotting and long-range PCR (data not shown). In a previous study, the promoter and transcribed sequences of H19 were sequenced in the DNAs of the patients BWS-16 and BWS-19 and no deletion or other mutation apart normal polymorphisms was found (29). We have now analysed the H19 promoter region by Southern blotting and sequenced the parts of the H19 transcribed region corresponding to known SNPs in all other patients. The normal pattern of bands detected by Southern blotting in all cases and the finding that most patients were heterozygous for at least one H19 intragenic polymorphic sequence variant excludes the presence of large H19 gene and promoter deletions in most cases (data not shown). Furthermore, neither mutation of the CDKN1C gene apart from normal polymorphisms (30) nor methylation defects at IC2 or 11p15.5 UPD were found in these patients (data not shown). All these cases were sporadic. The clinical features of these individuals and those carrying IC1 deletions are reported in Table 1. Overall, the phenotypes of these two groups of patients are very similar, both including the characteristics more often found in the BWS patients with defect of the IGF2-H19 imprinting domain, such as pronounced macrosomia, mild or absent defects of the abdominal wall and elevated incidence of WT (18).


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  		Table 1. Clinical features of the BWS patients with IC1 hypermethylation

 
Since IC1 deletions were present in 2/6 of the WTs developed in our BWS patients (Table 2), we looked for the presence of such mutations in sporadic cases of WT without other features of BWS. Consistent with the high frequency of 11p15.5 imprinting defects reported in these neoplasms, 10/40 of these tumours had IC1 hypermethylation by methylation restriction-PCR assay (MR-PCR or COBRA; Table 2). However, no IC1 deletion was detected in any of the 40 tumour cases by long-range PCR (Table 2).


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  		Table 2. Molecular defects at 11p15.5 in syndromic and non-syndromic WTs

 
Sequence analysis of IC1
The human IC1 region has a repetitive structure and contains seven target sites for CTCF (CTS) in 4 kb, with the first six CTSs arranged in two clusters of three sites each (Fig. 1A). In order to identify the cause of the epigenetic defect in the patients with IC1 hypermethylation and no deletion detected by Southern blotting, we searched for very small deletions or point mutations by sequencing the entire 4 kb region. However, no deletion was found in any of the 12 patients by this procedure. Some single-nucleotide variations with respect to the reference sequence (AF125183 [GenBank] ) were detected, but most of them corresponded to polymorphisms also found in the normal population. Indeed, common SNPs are present in three of the seven target sequences for CTCF (rs10840167, rs11042170, rs10732516). Few novel base substitutions were identified but none of them was de novo or present in more than one patient (Table 3). Since most of the patients were heterozygous for at least one SNP, the presence of larger deletions of the IC1 region was also excluded. It is therefore unlikely that the IC1 hypermethylation found in these patients is caused by a mutation in this region.


Figure 1
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  		Figure 1. Methylation of the CTSs and H19 promoter and IGF2 and H19 expression in the BWS patients with IC1 hypermethylation and no associated deletion.

(A) Summary of the results obtained on 12 BWS patients and 40 control individuals. DNA methylation at CpGs included in the CTSs and H19 promoter was assayed by MR-PCR (COBRA) in leukocyte DNA. The average methylation levels detected in control individuals (50±5%) are shown for comparison. The extent of methylation at each CpG site is indicated with pie charts filled in black. Each sample was run in duplicate. SD was ~5%. A diagram showing the structure of the human IC1 region is present in the upper part of the figure. A- and B-type repeats are indicated by boxes of different colours, whereas the CTSs are indicated by triangles. (B) DNA methylation profiles of CTCF site 1 and CTCF site 6 and flanking regions in patients BWS-19 and BWS-12 and a control individual, as determined by bisulfite-genomic sequencing. Each line corresponds to a single template DNA molecule cloned; each circle corresponds to a CpG dinucleotide. Filled circles designate the methylated cytosines; open circles, unmethylated cytosines. The CpGs included in the CTCF sites are framed. When indicated, the parental alleles were discriminated by SNPs. (C) Analysis of IGF2 and H19 expression in patient BWS-12. RNAs extracted from cultured skin fibroblasts of the patient and a control individual were analysed by reverse transcriptase–PCR. In the upper panel, the parental alleles of IGF2 were discriminated by typing for a direct repeat polymorphism (Can repeat) present in the 3'-untraslated region of the IGF2 gene. Three different alleles were observed. Both the maternal and paternal IGF2 alleles (a and b) were expressed in the fibroblasts of the patient, but only the paternal allele (b) was expressed in the control. To exclude contamination from DNA, the RNAs were amplified with (RT+) and without (RT–) previous treatment with reverse transcriptase. In the lower panel, the overall H19 RNA was determined by quantitative real-time RT–PCR using primers specific for the H19 transcript and the GAPD mRNA. Note that in the BWS patients without IC1 deletion the hypermethylation is generally mosaic and affects predominantly the CTSs that are closer to the H19 promoter. Regardless of the extension of the IC1 methylation defect, all these cases also have hypermethylation of H19 and several of them have been demonstrated to express IGF2 from both parental alleles and have H19 silenced.

 


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  		Table 3. Sequence variations of the IC1 region

 
DNA methylation and imprinting analysis
The analysis of methylation by Southern blotting does not provide information on the methylation status of the CTSs of the IC1 region. We have previously used MR-PCR or COBRA to demonstrate that the maternal transmission of IC1 deletions is associated with hypermethylation at all the remaining CTSs and the H19 promoter while 50% methylation is present in control individuals (17). We now used this procedure to analyse the 12 patients with hypermethylation at IC1 and no accompanying deletion (Fig. 1A). The results obtained showed differences in the extent of methylation of the individual CTSs and H19 promoter in these patients. Methylation was analysed in the peripheral blood leukocytes and ranged from 50 to 99% at different sites and in different patients. In addition, the degree of methylation was more homogeneous among the CTSs belonging to the same cluster and the CTSs 4–7 and H19 promoter were generally more methylated than the CTSs 1–3. Three individuals (BWS-12, BWS-19 and BWS-21) showed this characteristic in a more pronounced manner, having 50–60% methylation at the first three CTSs and 76–90% methylation at the further four CTSs and H19 promoter. The bisulfite-sequencing analysis confirmed the contrasting methylation between the 5' and 3' part of IC1 in these patients (Fig. 1B). DNA derived from the skin fibroblasts was available from patient BWS-12 and the MR-PCR demonstrated 80–90% methylation at the CTSs 4–6 and 50–57% methylation at the CTSs 1–3 also in this tissue (data not shown). We also analysed IC1 and H19 methylation at the CTSs and H19 promoter in the parents of the patients without IC1 deletion and in nine of their unaffected sibs and found methylation levels comparable to control individuals (data not shown). We then asked whether the methylation abnormality was specific for the IC1 or was extended to other imprinted loci. In addition to the 11p15.5 IC2, which had already been found normally methylated (see above), we analysed the DMRs of GTL2/DLK1, PEG3, ZAC and IGF2R by using MR-PCR (COBRA). We found ~50% methylation at all these loci in all our patients as well as in 50 control individuals (data not shown).

We have previously reported that the BWS patients with hypermethylation and silencing of H19 express IGF2 from both the maternal and paternal alleles (27,31). Two of these cases were further analysed in this manuscript (BWS-16 and BWS-19) and demonstrated to have the entire IC1 region or only the CTSs 4–7 hypermethylated, respectively (Fig. 1A). We now investigated the allele-specific expression of IGF2 and the overall expression of H19 in the RNA derived from the skin fibroblasts of the patient BWS-12. The results demonstrated expression from both parental IGF2 alleles, whereas expression from only the paternal allele was evident in the control, indicating loss of IGF2 imprinting also in this case (Fig. 1C). In addition, H19 RNA levels were 9-fold lower than in an age-matched control. No phenotypic difference was evident between the two groups of patients differing in the extension of the methylation defect at IC1. Overall, these results indicate that, in the BWS patients without IC1 deletion, the abnormal methylation extends over the entire IC1 region or affects mostly its 3' half. All these cases, regardless of the extension of the methylation defect in the IC1, also have hypermethylation (and probably silencing) of H19 and in several of them IGF2 has been demonstrated to be activated on both parental alleles. Furthermore, the methylation defect is specific for the 11p15.5 IC1 locus, is generally incomplete suggesting epigenetic mosaicism, and is not present in the unaffected relatives.

Segregation of 11p15.5 haplotypes
Since BWS results from an imprinting defect on the maternal chromosome, we analysed the segregation of the maternal IC1 allele of the index patient in the families without IC1 microdeletion after construction of 11p15.5 haplotypes by microsatellite analysis (Fig. 2A). In one family (BWS-20), the affected and an unaffected sib inherited the same 11p15.5 haplotype from their mother. In three additional families (BWS-11, BWS-17 and BWS-21), different 11p15.5 haplotypes were transmitted to the patients and their healthy siblings from their mothers. However, in family BWS-11, the propositus and her healthy mother shared the same 11p15.5 haplotype on the maternal chromosome. So, in at least two families, affected and non-affected individuals had the same maternal IC1 allele.


Figure 2
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  		Figure 2. Segregation of chromosome 11p15.5 genotypes in BWS families with IC1 imprinting defect and no associated deletion. (A) Construction of haplotypes by microsatellite analysis. The haplotype associated with the imprinting defect in the patient is framed. The chromosome location of the IC1 region is between TH and D11S1318. (B) Determination of the grandparental origin of the chromosome with the imprinting defect by combined DNA methylation/SNP test. The methylation of the IC1 region was analysed by bisulfite sequencing in the mothers of patients BWS-20, BWS-12 and BWS-14. Alleles were discriminated by SNPs and parental origin was assessed by assuming that the paternal IC1 allele was methylated and the maternal IC1 allele was not in normal individuals. DNA sequencing allowed to determine which maternal allele was transmitted to the probands. The SNPs and their positions in the AF125183 sequence are indicated. Note that affected and unaffected individuals share the same IC1 allele on the maternally inherited chromosome in some families and the chromosome with the imprinting defect in the probands derive from either the maternal grandfather or maternal grandmother.

 
The grandparental origin of the chromosomes carrying the imprinting defect was determined by microsatellite analysis (Fig. 2A) or (if grandparents were not available) DNA methylation/SNP test (Fig. 2B). In this procedure, the grandpaternal and grandmaternal IC1 alleles were distinguished in the DNA of the mothers because of their differential methylation (which was always all-or-none on the two parental alleles) determined by bisulfite sequencing. In case of heterozygosity, SNPs were used to determine which maternal allele was transmitted to the probands. Overall, the results showed that in five informative cases the chromosome carrying the imprinting alteration derived from the maternal grandfather, whereas in one case derived from the maternal grandmother (Table 4).


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  		Table 4. Grandparental origin of the chromosome carrying the imprinting defect

 
Mutation analysis of CTCF
Since CTCF binding is required for preventing de novo methylation at IC1 (32–35), we looked for mutations at the CTCF locus in the BWS patients with IC1 hypermethylation and no accompanying defect in cis. We sequenced all exons and flanking intronic regions in six of such patients. Only a single-nucleotide variation with respect to the reference sequence (NT_010498 [GenBank] , nucl. 21210663–21287287) was found in the 5'-UTR (Ex1 C/G19) of one individual (BWS-10) and an SNP also present in the normal population was found in the 3'-UTR (rs6499137) of another individual (BWS-12). No change in the coding sequence was present in any of these patients. Hemizygosity at the CTCF locus was also excluded in all cases by analysing a microsatellite present in intron 7 (rs3223529). Therefore, these results indicate that CTCF gene mutations are not a common cause of IC1 hypermethylation in BWS.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 ELECTRONIC-DATABASE INFORMATION
 FUNDING
 REFERENCES
 
Gain of methylation defects at IC1 are found in only 5–10% of the BWS cases (12,18). However, the high incidence of WT associated with these molecular abnormalities makes them particularly important to study. Some of the patients with this type of abnormal methylation have been demonstrated to have germline deletions in the IC1 region and high recurrence risk (13,17). Other BWS patients with similar epigenetic defect at IC1 had no associated deletion. It was unclear whether these patients had any other mutation in cis, what was the nature of their molecular defect and what was their recurrence risk. By analysing 12 patients with IC1 hypermethylation and no deletion, we found no mutation in the critical IC1 elements and demonstrated that, in at least two cases, the maternal IC1 allele of the index patient segregated in one of his healthy relatives. A detailed methylation analysis showed that the hypermethylation was extended over the entire or only the 3' half of the IC1 region, did not affect other imprinted loci, generally occurred in the mosaic form and was never present in the unaffected relatives. We also observed that the chromosome carrying the imprinting abnormality derived from either the maternal grandfather or maternal grandmother. These results indicate that, in the absence of deletions, IC1 hypermethylation generally occurs as sporadic epimutation and is associated with low recurrence risk. Consistent with this conclusion, no deletion was detected in a large series of non-syndromic sporadic WTs.

In the mouse, the CTSs have been shown to be required for the post-zygotic maintenance of the methylation-free status of the maternal IC1 allele (32–35). Consistent with these observations, deletions removing one to two CTSs of the human IC1 result in the hypermethylation of its maternal allele (13,17). However, we found no mutation in any of the seven CTSs of another 12 BWS patients with IC1 hypermethylation. In addition, the segregation of the chromosome with the imprinting defect in the healthy relatives excludes inherited mutations in the entire chromosome 11p15.5 region of two individuals. Therefore, although we cannot rule out the presence of de novo mutations at sites distant from IC1, our results strongly support the hypothesis that in a subset of BWS patients IC1 epimutations occurr independently of the sequence context.

We previously observed that all IC1 CTSs are completely and exclusively methylated on the paternal chromosome in normal leukocyte DNA, whereas incomplete hypermethylation of the maternal allele is present in the BWS patients with 1.4–1.8 kb IC1deletions (17). We now found incomplete methylation at IC1 also in the majority of patients without deletions suggesting that this imprinting defect is generally present in the mosaic form. Mosaicism can in part explain the high variability of the clinical phenotype that is characteristic of this disorder. In addition, diagnostic problems may be encountered with individuals who may not have abnormal methylation in their leukocyte DNA. To avoid these problems, it may be useful in the future to analyse DNAs derived from more than one tissue (e.g. blood leukocytes and buccal mucosa).

Imprinting defects at ICs can derive from failure of erasure, establishment or maintenance of the parental marks (8). One way to approach this problem is to investigate the grandparental origin of the chromosome with the imprinting defect (36). We found that in the BWS patients the chromosome with abnormal IC1 methylation derived from either the maternal grandfather or the maternal grandmother. Since the majority of these cases also showed mosaic hypermethylation, it is likely that the methylation defect was acquired after erasure of the parental epigenetic marks and probably at a post-zygotic stage as result of defective protection from de novo methylation of the maternal IC1 allele. A similar mechanism is also likely to be involved in the cases with IC1 deletion (17). However, an incomplete imprint erasure resulting in an unstable methylation is also possible, at least in some cases.

The ultimate effect of IC1 methylation on the maternal chromosome is H19 silencing (by methylation spreading into the promoter region) and IGF2 activation [by inactivation of the insulator activity (8)]. We found that in some of the BWS patients hypermethylation is more evident on the four CTSs that are located closer to the H19 promoter. This may indicate that methylation of the CTSs 4–7 is sufficient to cause silencing of H19 and/or activation of IGF2. Indeed, gain of methylation at the H19 promoter was observed in all the patients regardless of the extension of IC1 hypermethylation and biallelic activation of IGF2 could be demonstrated in two of the patients with limited IC1 methylation. Cooperative interaction between proteins bound to adjacent CTSs (33) may explain the more homogeneous methylation inside each cluster of CTSs.

Since we did not find a mutation in cis, it is possible that a regulatory trans-acting factor is defective in the BWS cases with IC1 hypermethylation and no deletion. Knocking-down of CTCF in mouse oocytes and in cultured cells results in gain of methylation of IC1 (7,37). We ruled out the presence of a mutation of the CTCF gene in our patients. It cannot be excluded, however, that a mutation is present in other modifier genes. Considering the sporadic nature of these cases, the possibility that IC1 hypermethylation occurs as consequence of stochastic events or environmental influence should also be envisaged (38).

The 11p15.5 imprinted gene cluster is frequently affected in WTs (28). Either maternal deletion/paternal duplication (LOH) or IC1 hypermethylation coupled to H19 silencing and IGF2 activation (LOI) can be found in a high proportion of tumours (see also Table 2). In addition, the individuals who have soma-wide IC1 hypermethylation or 11p15.5 paternal UPD represent the molecular subgroup of BWS patients showing the highest risk of developing WT (18–20). Consistent with these observations, we found that 2/6 BWS patients with IC1 deletion had developed this neoplasm by 6 years of age. However, no IC1 deletion was found in 40 sporadic non-syndromic WTs enrolled by Pediatric Oncology Units affiliated to Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP). A previous American study demonstrated the absence of point mutations in the IC1 sequence of a series of sporadic WTs (39). Overall, these data suggest that IC1 imprinting defects are generally not caused by a mutation in cis in non-syndromic WT as well as sporadic BWS.

Although the common hallmark and probably the ultimate cause of the imprinting defects at the IGF2/H19 locus is represented by hypermethylation of IC1, our studies demonstrate that this epigenetic abnormality can result from more than one mechanism in BWS and WT. In a first group of patients, we found that the epimutation is a direct consequence of a mutation in cis, consisting of a deletion of one to two CTSs. In these cases, the methylation defect and disease phenotype are reproduced whenever the mutation is transmitted through the maternal germline. In another group of patients, who carry no IC1 deletion, the epimutation is independent of the local DNA sequence and generally not transmitted to the progeny. Sporadic BWS and non-syndromic WTs belong to the second group of cases. Despite these differences, the IC1 epimutation is generally present in the patients in the mosaic form and probably acquired by postzygotic de novo methylation, providing an example of how intricate the relationship between genotype and epigenotype can be.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
ELECTRONIC-DATABASE INFORMATION
 FUNDING
 REFERENCES
 
Patients
BWS was diagnosed in all patients according to the criteria described in the literature (http://www.geneclinics.org). The clinical features of the BWS patients included in this study are summarized in Table 1. The study included 40 WT patients enrolled in the AIEOP 2003 WT trial. In 37 of these cases, there was no evidence of associated congenital abnormalities. The three other cases were associated with WAGR, Turner and Down syndrome phenotypes, respectively.

All the genetic analyses were performed after the informed consent was obtained from the parents of the patients. The experimental plan was approved by the ethical committees of the Second University of Naples and Istituto Nazionale Tumori, INT, Milan.

Southern blot, PCR, DNA sequencing and microsatellite analyses
Southern blot hybridization was performed on DNA purified from blood leukocytes as described (13,31). PCR amplification of the H19 DMR was obtained from leukocyte DNA by using the primers 5'-AGAGATGGGATTTCGTCAGGTTGG-3' and 5'- CATTTCCGTCTCCACAGCCACAAC-3' and the Taq BIO-X-ACT Long (BIOLINE) as described (13). The fragments generated were gel-purified and cloned into pCR II (Invitrogen). The primers used for sequencing the H19 and CTCF gene and the promoter, 5'-UTR and 3'-UTR of CDKN1C are reported in Table 5. The primers used for sequencing CDKN1C exons 3–4 were previously described (40). DNA sequencing was obtained from PRIMM (Italy). UPD and LOH at 11p15.5 loci were determined by microsatellite analysis, as described (18).


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  		Table 5. PCR primers used for the sequence analysis

 
DNA methylation analyses
The DNA methylation of IC1 (H19 DMR) and H19 promoter was analysed by HpaII digestion and Southern blot hybridization, sodium bisulfite sequencing and bisulfite treatment coupled with restriction enzyme digestion (MR-PCR), as described (13). The methylation of IC2 (KvDMR1) was analysed by NotI digestion and Southern blotting hybridization, as described (18). The DNA methylation of the DMRs of ZAC, IGF2R, PEG3 and GTL2/DLK1 were analysed by MR-PCR. The PCR conditions and the enzymes used for each DMR are described in Table 6.


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  		Table 6. MR-PCR conditions for the methylation analysis of the DMRs at ZAC, IGF2R, PEG3 and GTL2/DLK1 loci

 
Gene expression analysis
The allele-specific expression of IGF2 and the overall expression of H19 were analysed as previously described (13).


   ELECTRONIC-DATABASE INFORMATION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 ELECTRONIC-DATABASE INFORMATION
FUNDING
 REFERENCES
 
URLs for data in this article are as follows:

Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim [for BWS (MIM 130650 [OMIM] )]. http://www.mgu.har.mrc.ac.uk/research/imprinting/;http://igc.otago.ac.nz/home.html; http://www.geneclinics.org


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 ELECTRONIC-DATABASE INFORMATION
 FUNDING
REFERENCES
 
This work was supported by grants from MIUR PRIN 2005 (to A.R. and M.V.C), Istituto Superiore di Sanità (to A.R.), Associazione Italiana Ricerca sul Cancro (to A.R. and D.P.), Telethon-Italia Grant No. GGP04072 (to A.R.), Associazione Bianca Garavaglia, Busto Arsizio, Varese, Italy (to D.P.) and WellChild (to E.R.M.). F.C. was recipient of a fellowship from Società Italiana di Cancerologia and Fondazione Pezcoller. A.S. was supported by a fellowship from the Regional Competence Center on Genomics (GEAR) of Regione Campania.


   ACKNOWLEDGEMENTS
 
We thank the contribution of the AIEOP Wilms Tumor Scientific Committee, all the patients and their families for their participation in this study.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 ELECTRONIC-DATABASE INFORMATION
 FUNDING
 REFERENCES
 

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