Human Molecular Genetics, 2001, Vol. 10, No. 26 2989-3000
© 2001 Oxford University Press
Tumor development in the Beckwith–Wiedemann syndrome is associated with a variety of constitutional molecular 11p15 alterations including imprinting defects of KCNQ1OT1
1Division of Clinical and Metabolic Genetics and the Department of Paediatrics and 2Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada, 3Department of Molecular and Medical Genetics and 4Institute of Medical Sciences, University of Toronto, Toronto, Ontario, Canada, 5Department of Paediatric Laboratory Medicine, Hospital for Sick Children, Toronto, Ontario, Canada, 6Ontario Cancer Institute, Toronto, Ontario, Canada, 7Department of Laboratory Medicine and Pathobiology and 8Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
Received August 10, 2001; Revised and Accepted October 29, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Dysregulation of imprinted genes on human chromosome 11p15 has been implicated in Beckwith–Wiedemann syndrome (BWS), an overgrowth syndrome associated with congenital malformations and tumor predisposition. The molecular basis of BWS is complex and heterogeneous. The syndrome is associated with alterations in two distinct imprinting domains on 11p15: a telomeric domain containing the H19 and IGF2 genes and a centromeric domain including the KCNQ1OT1 and CDKNIC genes. It has been postulated that disorders of imprinting in the telomeric domain are associated with overgrowth and cancer predisposition, whereas those in the centromeric domain involve malformations but not tumor development. In this study of 125 BWS cases, we confirm the association of tumors with constitutional defects in the 11p15 telomeric domain; six of 21 BWS cases with uniparental disomy (UPD) of 11p15 developed tumors and one of three of the rare BWS subtype with hypermethylation of the H19 gene developed tumors. Most importantly, we find that five of 32 individuals with BWS and imprinting defects in the centromeric domain developed embryonal tumors. Furthermore, the type of tumors observed in BWS cases with telomeric defects are different from those seen in BWS cases with defects limited to the centromeric domain. Whereas Wilms’ tumor was the most frequent tumor seen in BWS cases with UPD for 11p15 or H19 hypermethylation, none of the embryonal tumors with imprinting defects at KCNQ1OT1 was a Wilms’ tumor. This suggests that distinct tumor predisposition profiles result from dysregulation of the telomeric domain versus the centromeric domain and that these imprinting defects activate distinct genetic pathways for embryonal tumorigenesis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Beckwith–Wiedemann syndrome (BWS) (OMIM 130650; http://www3.ncbi.nlm.nih.gov/htbin-post/Omim/dispmim?130650) is a phenotypically and genotypically heterogeneous overgrowth syndrome (1–4). The most common phenotypes associated with BWS are pre- and post-natal overgrowth, organomegaly, hemihyperplasia, omphalocele, ear lobe and renal abnormalities. Children with BWS have a 7–21% risk for the development of embryonal malignancies, most notably Wilms’ tumor of the kidney (5–8). However, a wide variety of benign and malignant tumors have been reported (1,5). These malignancies include hepatoblastoma, adrenocortical carcinoma, rhabdomyosarcoma and neuroblastoma (5).
BWS has been associated with a diversity of genetic and epigenetic alterations on chromosome 11p15 (3,4). The imprinted gene cluster on human chromosome 11p15.5 has been proposed to consist of two domains (3,4,9) (Fig. 1). The telomeric domain includes the paternally expressed gene IGF2 (insulin-like growth factor 2) and the maternally expressed gene H19, whereas the centromeric domain includes the maternally expressed genes CDKNIC (p57KIP2), KCNQ1 (KvLQT1) and the paternally expressed gene KCNQ1OT1 (KvLQT1-AS, LIT1). Two non-imprinted genes TSSC4 and PHEMX (10) map to the 11p15 region between the two imprinted gene clusters.
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In the telomeric domain, H19 encodes an apparently untranslated RNA (11). The function of this gene is presently unresolved but it is involved in regulating the expression of the IGF2 gene, which encodes a fetal growth factor. A putative imprinting control center, named H19DMR, is located upstream of H19, and regulates H19 and IGF2 expression.
On the expressed maternal H19 allele, H19DMR is non-methylated and is able to bind the CTCF protein. This insulates the maternal IGF2 promoter from two enhancers located 3' of the H19 gene and prevents expression of IGF2. On the paternal allele, the H19 DMR is methylated. This not only prevents expression of the imprinted paternal H19 alleles, but it also blocks the binding of the CTCF protein (12). The IGF2 promoter now has access to the 3' enhancers and the paternal allele is expressed via enhancers and boundary elements in the region (13,14).
In the centromeric domain, CDKNIC encodes an inhibitor of cyclin-dependent kinases and is involved in cell cycle regulation. KCNQ1 encodes a voltage-gated potassium channel. The KCNQ1OT1 gene encodes a paternally expressed, untranslated transcript which is read in antisense orientation to and overlapping with the KCNQ1 gene (9,15). KvDMR1 is a maternally methylated CpG island at the 5' end of the KCNQ1OT1 transcript (9,15,16). Recently, Horike et al. (17) generated modified human chromosomes carrying targeted deletions of KvDMR1. In these cells the KCNQ1OT1 gene was silenced and the KCNQ1 and CDKNIC genes were overexpressed from the paternal chromosome bearing the deletion. These results suggest that KvDMR1 may function as a regional imprinting center (BWSIC2) that regulates the centromeric 11p15 imprinted domain.
The genetics of BWS is complex. Approximately 15% of cases are familial and 1–2% have translocations and duplications of 11p15 (1,2). The majority (
85%) of BWS cases are sporadic with normal karyotypes. Of the sporadic cases, 20% exhibit somatic mosaicism for paternal uniparental disomy (UPD) of both the centromeric and telomeric domains (18,19). Sporadic cases of BWS without 11p15 UPD exhibit changes in a number of imprinted genes, including mutations in the CDKNIC gene (5% of sporadic cases) (20,21) and loss of imprinting of IGF2 resulting in biallelic expression (22). This loss of imprinting of IGF2 can occur in two situations: rarely in conjunction with hypermethylation of H19 in 1–2% of BWS cases (23), or more commonly, accompanied by normal methylation of the H19 gene (22,24). Recently, it has been shown that 50% of sporadic BWS cases show loss of methylation at KvDMR1 (9,15,25,26). Lee et al. (9) found that loss of methylation at KvDMR1 was associated with biallelic expression of KCNQ1OT1 in eight of 16 BWS cases.
Several studies have shown that dysregulation of imprinting in the telomeric domain is associated with predisposition to tumors in BWS (27). Lee et al. (9) suggested that the two 11p15 imprinting domains have overlapping but non-identical functions in BWS, with the telomeric domain primarily involved in cancer predisposition. Moreover, Tycko (27) proposed that H19 hypermethylation was strongly associated with tumorigenesis either in the context of 11p15 UPD or as an isolated epigenetic event. These findings were supported by reports of cases of BWS and tumors associated with 11p15 UPD (25,26) and with the rare epigenotype of hypermethylation of H19 (25,27). However, mutation of CDKNIC, a maternally expressed candidate tumor suppressor gene located in the centromeric imprinting domain, has been associated with tumor development in only two BWS cases to date (28,29). No information regarding tumor status was reported for 30 cases of BWS with imprinting defects of KvDMR1 (9,15,16). In addition, there have been only two studies of BWS patients with loss of methylation at KvDMR1 which reported no embryonal tumors (25,26). Recently, only one malignant tumor was reported for a non-UPD BWS patient with demethylation at KvDMR1 (29). Thus, to date, only two BWS molecular subgroups have been clearly implicated in the tumor predisposition associated with BWS, i.e. UPD of 11p15 and hypermethylation of H19 (30). Heretofore, imprinting defects at the KCNQ1OT1/KvDMR1 locus have not been considered to predispose to embryonal tumor development in BWS (25,26).
In this work, we examine the molecular lesions that occur in cases of BWS with tumors. We confirm that 11p15 UPD and H19 hypermethylation are indeed associated with tumor development. We demonstrate that BWS patients with imprinting defects at KCNQ1OT1/KvDMR1 are also at risk for tumor development. Furthermore, the data suggest that the types of tumors found in BWS cases with constitutional imprinting defects involving the telomeric domain are distinct from those associated with disruption of imprinting of the centromeric domain.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Chromosome 11 uniparental disomy
Analysis for UPD of chromosome 11 was carried out using quantitative multiplex-PCR with highly polymorphic STR markers. At least two informative markers in the 11p15 region were required to complete the analysis. Normal bi-parental inheritance of these 11p15 markers was found in all 12 control samples (Fig. 2A). Of 125 BWS cases tested, we detected 21 cases of UPD for 11p15 (Fig. 2B). In 17 of these cases, analysis of parental samples confirmed paternal origin of the UPD. In the other four cases, parental samples were not available. For all 21 cases of 11p15 UPD, somatic mosaicism was evident, the percentage varying from 25 to 95%. In fact, for two cases of BWS, 11p15 UPD was detected in only some of the available tissue samples. Figure 2C shows data for one such individual with hemihyperplasia affecting one whole side of the body. The skin fibroblast cells derived from the arm with hemihyperplasia showed UPD for 11p15 whereas the fibroblast strain from the other arm and from lymphocytes showed normal biparental inheritance of 11p15 markers. Six (29%) of the 21 cases of UPD developed malignant tumors. These included five Wilms’ tumors and one hepatoblastoma.
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CDKNIC mutations
DNA from BWS patients was screened for coding mutations in the CDKNIC gene. Mutations were identified in five patients of the 125 patients screened. All mutations were inherited from the mother except in one case where paternal transmission was noted. Four of the five mutations have been previously published (31). The new mutation was identified in a sporadic BWS case and is a C
T transition at nucleotide 432. This change results in a single amino acid substitution at residue 58 converting a proline (CCG) to a leucine (CTG) in the CdK inhibitory domain (P58L mutation).
Methylation status of H19
In order to assess the methylation status of the H19 gene, we analyzed the differentially methylated SmaI site near the promoter of H19 by Southern blot analysis. A methylation index (MI) was calculated as formulated by Reik et al. (32). For 10 controls (Fig. 3A), MIs gave a mean of 0.50 with a SD of ±0.05. We defined hypermethylation at the H19 locus as a MI of 
0.60 (mean + 2SD). We found hypermethylation of the H19 gene in 24 of 125 BWS cases. Of these, 21 cases had UPD (Fig. 3B), leaving three of 125 cases with hypermethylation of H19 as an independent finding (Fig. 3C). All 12 familial cases showed a normal methylation pattern for H19 (data not shown). An anaplastic Wilms’ tumor was found in one (33%) of the three BWS cases with H19 hypermethylation.
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Methylation status of KvDMR1
The methylation status of KvDMR1 was analyzed using the differentially methylated NotI site in the CpG island upstream of KCNQ1OT1, KvDMR1. The MI was calculated as for H19. For 10 controls (Fig. 4A), a mean MI of 0.50 was obtained with a SD of ±0.04. A MI
0.42 (mean – 2SD) was defined as hypomethylation at KvDMR1.
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We analyzed 72 BWS cases for methylation at KvDMR1 methylation. As expected, all 13 patients (18%) with UPD exhibited hypomethylation at KvDMR1 (Fig. 4B). In addition, 35/59 (59%) of the remaining 59 non-UPD BWS cases exhibited hypomethylation at KvDMR1 (Fig. 4C and Table 1). In 11 cases, complete demethylation was observed; in 25 cases the demethylation was incomplete. We found malignant tumors in five (14%) of the 36 non-UPD BWS cases with hypomethylation at KvDMR1.
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Expression analysis of KCNQ1OT1
In order to evaluate whether hypomethylation at KvDMR1 was consistently associated with altered expression of the imprinted transcript KCNQ1OT1 we assayed its expression using PCR-based assays of three polymorphic SNPs (9). All 12 controls, informative for at least one SNP, showed monoallelic expression of KCNQ1OT1 (Fig. 5A and Table 1). This finding is consistent with the normal MIs obtained for KvDMR1 of 0.48–0.53. We found at least one informative SNP in KCNQ1OT1 in 67 of 72 BWS cases. Of these 67 cases, 35 (52%) showed biallelic expression of KCNQ1OT1 (Fig. 5B and Table 1). These 35 cases all demonstrated hypomethylation of KvDMR1 with MIs ranging from 0 to 0.33. The remaining 32 BWS cases showed monoallelic expression of KCNQ1OT1 transcript (Fig. 5C and Table 1), including two cases with hypermethylation of the H19 gene (Fig. 5D and Table 1). Informative parental samples available in 24 cases demonstrated that this monoallelic expression was of paternal origin. For 19 of the 32 BWS cases with monoallelic expression of the KCNQ1OT1 transcript, we observed normal methylation at KvDMR1 (MIs of 0.46–0.53). The remaining 13 cases all had UPD for 11p15 and showed hypomethylation at KvDMR1 (MIs of 0.35–0.10) (Fig. 5E and Table 1). That is, apparent discordance between methylation status at KvDMR1 and transcription of KCNQ1OT1 is a hallmark of BWS cases with UPD. This profile reflects the differential parental contributions in the 11p15 region in the absence of a primary imprinting error.
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Combined molecular analysis of 11p15
We have completed molecular analysis for UPD of 11p15, methylation of the H19 gene and CDKNIC mutations in 125 cases of BWS. Methylation at KvDMR1 and allelic transcription of KCNQ1OT1 were assessed in 72 BWS cases. From these data, we have grouped the patients into five molecular classes: group I, UPD of 11p15; group II, methylation errors at H19; group III, methylation/transcription errors at KvDMR1/KCNQ1OT1; group IV, CDKNIC mutations; group V, normal methylation patterns of KvDMR and H19. A summary of the results is listed in Table 2. There were no statistical differences in tumor frequencies among groups I, II, III and IV.
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Delineation of BWS patient molecular subgroups
Group I: UPD. This group is defined by (i) MI for H19 of
0.60, (ii) MI for KvDMR1 of 0.42, (iii) monoallelic expression of KCNQ1OT1 and (iv) greater paternal than maternal contribution of 11p15 alleles.
We assigned 21 of 125 BWS cases to the UPD group based on a greater paternal than maternal 11p15 allelic contribution as well as a MI for H19 of 0.60. Thirteen of the UPD cases were available for testing for KvDMR1. All of these showed a MI for KvDMR1 of 0.42 and normal monoallelic expression of KCNQ1OT1. Six of the 21 UPD cases developed tumors, including five Wilms’ tumors and one hepatoblastoma (Table 2).
Group II: methylation error of H19. This group is defined by (i) MI at H19 of 0.60, (ii) MI at KvDMR1 of 0.50 ± 2SD, (iii) monoallelic expression of KCNQ1OT1 and (iv) normal biparental 11p15 allelic contributions. Three of 125 (2%) had a MI at H19 of 0.60–0.72, 0.78 and 0.84 with corresponding MIs at KvDMR1 of 0.52, 0.54 and 0.49, respectively. All three showed monoallelic expression of KCNQ1OT1 and normal biparental inheritance of 11p15 markers. One (33%) of these three patients developed a Wilms’ tumor.
Group III: methylation error of KvDMR1 and biallelic expression of KCNQ1OT1. This group is defined by (i) MI at H19 of 0.50 ± 2SD, (ii) MI at KvDMR1 of 0.42, (iii) biallelic expression of KCNQ1OT1 and (iv) normal biparental 11p15 allelic contributions.
Thirty-five (59%) of 59 BWS cases (Table 2) showed normal biparental inheritance of 11p15 markers and hypomethylation at KvDMR1 with concomitant biallelic expression of KCNQ1OT1. All 35 showed normal methylation at H19 (MIs of 0.46–0.55), supporting the hypothesis that H19 and KvDMR1 methylation errors occur independently. Of the 35 BWS cases in this group, five (14%) developed tumors. Surprisingly, none of these was a Wilms’ tumor. The tumors included two hepatoblastomas, two rhabdomyosarcomas and one gonadoblastoma. These five BWS patients have normal biparental inheritance of 11p15 markers (data not shown) and normal H19 MIs (Fig. 3D–H), thereby excluding mosaicism for 11p15 UPD and defects of H19 methylation. However, all five cases show loss of maternal methylation at KvDMR1 (Fig. 6A–E). Furthermore, allelic expression studies of KCNQ1OT1 are available for three of these five cases and all show biallelic expression of KCNQ1OT1 (Fig. 6F–H). Therefore, the constitutional molecular defects in these five cases of BWS with non-Wilms’ embryonal tumors must involve primary epigenetic alterations at KvDMR1/KCNQ1OT1.
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Group IV: CDKNIC mutations. This group is defined by (i) MI at H19 of 0.50 ± 2SD, (ii) MI at KvDMR1 of 0.50 ± 2SD, (iii) monoallelic expression of KCNQ1OT1, (iv) normal biparental 11p15 allelic contributions and (v) mutations in CDKNIC.
Five of 125 BWS cases had mutations in CDKNIC. All five had normal molecular analyses for H19 and KvDMR1 methylation, monoallelic expression of KCNQ1OT1 and normal biparental 11p15 allelic contributions. None of these BWS cases had embryonal tumors.
Group V: normal methylation. This group is defined by (i) MI at H19 of 0.50 ± 2SD, (ii) MI at KvDMR1 of 0.50 ± 2SD, (iii) monoallelic expression of KCNQ1OT1 and (iv) normal biparental 11p15 allelic contributions. Seventeen (25%) of 67 BWS cases (Table 2) showed normal biparental 11p15 contributions with normal methylation at H19 and KvDMR1 accompanied by monoallelic expression of KCNQ1OT1. Four (24%) of 17 had Wilms’ tumors. These four cases all had classical features of BWS including macrosomia, macroglossia and hemihyperplasia and none of these cases was familial.
Comparison of our data to published data on tumor risk and BWS molecular subgroups
Table 3 compares our data to the published data for tumor risk associated with specific BWS molecular subgroups in the telomeric and centromeric domains. Our data found six (29%) tumors in 21 cases of BWS with 11p15 UPD, consistent with that previously reported by several other groups (Table 3). All of these studies attribute tumor development in BWS cases either to UPD of 11p15 or methylation defects at H19. Bliek et al. (25) found four (18%) tumors among 22 cases of BWS with UPD of 11p15. Both Engel et al. (26) and Gaston et al. (29) compiled previous and new data reporting two (9%) tumors in 22 BWS cases with 11p15 UPD and four (36%) in 11 cases of BWS with 11p15 UPD, respectively. For the BWS patients with H19 methylation errors, although only small numbers are available, our findings (one of three) are consistent with those previously reported (five of 14) in that patients in this molecular subgroup are at increased risk to develop tumors (Table 3). Overall, the data highlight the significant tumor risk associated with molecular defects in the telomeric domain. Combining our data with those in the literature, 25 tumors have been observed in the 107 BWS cases with telomeric molecular defects and a tumor incidence of 24%.
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In the centromeric domain, although we did not find any tumors in five cases of BWS with CDKNIC mutations, there are two neuroblastomas in approximately 33 BWS individuals with such mutations. Overall, the tumor incidence for CDKNIC mutation is 2/38 or 5.2% (Table 3).
Of particular interest are the data on tumor incidence in BWS cases with methylation and transcription errors at KvDMR1 and KCNQ1OT1. Our results showing five tumors in 35 BWS cases with imprinting defects at KvDMR1/KCNQ1OT1 are statistically different from those previously reported for BWS cases with such constitutional alterations (Fisher test P < 0.01). Engel et al. (26) and Bliek et al. (25) each reported no tumors in 29 cases of BWS with methylation errors at KvDMR1. One recent study (29) identified a single tumor in 30 cases of BWS with KvDMR1 methylation defects which further supports our new findings. Thus, to date, of 123 BWS cases reported with imprinting defects at KCNQ1OT1, six (5%) have developed a malignant tumor.
A review of the tumor types associated with specific molecular subgroups of BWS demonstrates clear differences (Table 4). Our data show that whereas Wilms’ tumor is the common tumor seen in BWS cases with molecular defects in the telomeric domain, it is not represented in the five tumors observed in BWS cases with imprinting errors at KCNQ1OT1 (Table 4). Instead, we see a variety of non-Wilms’ tumors including rhabdomyosarcoma and gonadoblastoma. Hepatoblastoma is seen with molecular defects in both domains. A review of such data from the literature further supports our finding. Combining all the available data for BWS cases with known molecular defects and tumors (Table 4) it appears that for 11p15 UPD and methylation errors at H19, 22 (88%) of 25 tumors reported are Wilms’ tumors. The other three tumors are hepatoblastoma, neuroblastoma and pheochromocytoma. However, in the centromeric domain of the eight tumors reported to date none is a Wilms’ tumor (Table 4). The present study demonstrates two BWS cases with rhabdomyosarcomas, two BWS cases with hepatoblastomas, and one BWS case with gonadoblastoma and an imprinting defect at KCNQ1OT1. In addition, one other group has reported a thyroid carcinoma in a BWS case with an imprinting defect at KCNQ1OT1 (29). However, since thyroid carcinoma has not previously been reported in BWS, it is not clear whether the thyroid carcinoma occurred by chance or is truly associated with the imprinting defect at KCNQ1OT1. For CDKNIC mutations, also in the centromeric domain, two cases of neuroblastoma have been reported (28,29).
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For the group of BWS cases in which the tests including UPD analysis, H19 methylation KCNQ1OT1, and CDKNIC mutation analysis are negative (group V), the rate of tumors is six (22%) of 27 cases. In addition, all six of these group V patients, four in our study and two from Bliek et al. (25) had hemihyperplasia. The tumor type is Wilms’ tumor in all six cases, a profile suggestive of a telomeric domain molecular defect.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In this paper, we have shown that several constitutional BWS molecular defects are represented in BWS cases with tumors. We confirm that these molecular defects include UPD of 11p15 and biallelic methylation of the H19 gene. Most importantly, this is the first report of embryonal tumors in BWS cases with constitutional imprinting defects at KCNQ1OT1. Dysregulation of imprinting at KCNQ1OT1 and/or KvDMR1, was found to be independent of methylation of the maternal H19 gene or of UPD at 11p15. These findings expand on current molecular models of tumor predisposition on 11p15 (3,4,30), and suggest that both the telomeric and centromeric imprinted domains are implicated in embryonal tumorigenesis. Furthermore, our data suggest a molecular basis for the diversity of tumors observed in BWS. Our data imply that tumor risk associated with UPD of 11p15 and H19 methylation errors is different from that observed in BWS individuals with imprinting defects at KCNQ1OT1 and/or KvDMR1. Specifically, Wilms’ tumor is associated with BWS molecular defects in the telomeric domain, whereas rhabdomyosarcoma and gonadoblastoma are associated with molecular lesions in the centromeric domain. Hepatoblastoma occurs in BWS cases with molecular lesions in either domain.
11p15 UPD in BWS patients
BWS patients with UPD of 11p15 (group I) have an increased paternal genomic contribution. Because the majority of these cases exhibit somatic mosaicism, abnormal methylation analyses of genes in the 11p15 region will reflect the degree to which the paternal methylation contribution is over-represented. ‘Hypermethylation’ of H19 is observed, whereas ‘hypomethylation’ of KvDMR1 is seen. As previously noted by Bliek et al. (25), the MIs generated at each locus are skewed to the same extent, but in opposite directions by the increased contribution of paternal alleles. Thus, for cases of UPD of 11p15, the combined MIs for H19 and KvDMR1 approach unity. Furthermore, the degree of methylation distortion is proportional to the level of mosaicism in the tissue being analyzed. Whereas methylation assays for cases of UPD for 11p15 will be skewed, transcription assays should demonstrate the integrity of the cis-acting imprinting regulatory mechanisms of 11p15. As anticipated, we have shown in this work, despite hypomethylation at KvDMR1, monoallelic paternal transcription of KCNQ1OT1 is maintained in cases of 11p15 UPD. However, since we assayed the allelic contributions by PCR, we have not assessed quantitatively the steady-state levels of KCNQ1OT1 RNA. These may be elevated by the presence of UPD for 11p15 (Fig. 7).
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The frequency of 11p15 UPD in our series was 17%. This is in keeping with data obtained by other groups (18,19,25,26,29,33–36). However, our demonstration of two cases where UPD was found in skin fibroblasts from one side of the body but not the other suggests that 11p15 UPD may exhibit tissue specificity or mosaicism and be more frequent than the literature reports, since analysis is usually limited to blood.
The tumor risk (29%) associated with 11p15 UPD in our patient series is comparable to that reported by other investigators (Table 3). However, because the tumor types observed in our study group were more diverse than in most previous studies, we were able to demonstrate a specific association between 11p15 UPD and the development of Wilms’ tumor. This association between Wilms’ tumorigenesis and dysregulation of the 11p15 telomeric imprinted domain parallels a body of molecular data generated for sporadic Wilms’ tumor (30). That is, maternal-specific loss of heterozygosity in 11p15 has been observed often in Wilms’ and other embryonal tumors, involving a region of at least 800 kb that is proposed to harbor the WT2 tumor susceptibility gene (37). Specific patterns of acquired gene dysregulation in tumors associated with the development of sporadic Wilms’ tumor indicate a role for somatic dysregulation of the 11p15 imprinted telomeric domain during tumorigenesis (38).
H19 methylation errors in BWS patients
This group of BWS cases demonstrates methylation of the normally unmethylated maternal H19 gene as a result of an epigenetic alteration in BWSIC1, the putative imprinting center upstream of H19. Concomitant changes in allelic transcription have been reported, with loss of maternal H19 expression and biallelic IGF2 expression (30). In our study, one of three patients with this epigenotype developed a tumor, a finding supported by a series of studies (23,25,26,33). As for most of the 11p15 UPD cases, the tumors reported by others have been exclusively Wilms’ tumors. In fact, H19 hypermethylation is a well-established epigenotype associated with sporadic Wilms’ tumor. Usually, hypermethylation and decreased expression of the H19 gene with concomitant loss of imprinting of the maternal allele of the IGF2 gene are seen (30,39,40). Thus parallel changes in epigenotype occur in this class of BWS patients and in sporadic Wilms’ tumors.
Imprinting defects of KvDMR1/KCNQ1OT1 in BWS patients
The differentially methylated region (DMR), KvDMR1, has been postulated to represent BWSIC2, the imprinting center for domain 2. An imprinting defect at this locus results in loss of methylation of the maternal KvDMR1 region. To date, such cases with loss of maternal methylation at KvDMR1 have been shown to exhibit concomitant expression of the normally silenced maternal KCNQ1OT1 in only a small number (n = 8) of patients. In keeping with a model in which regulation of this centromeric imprinted domain is an independent process, aberrant imprinting at both KvDMR1 and KCNQ1OT1 was not accompanied by abnormal methylation of the H19 gene.
The frequency of KvDMR1 defects in our series of BWS patients is 61%, which is comparable to that in the literature. We found 11/36 cases with complete demethylation and 25/36 cases with partial demethylation. These data are consistent with those of Bliek et al. (25). Moreover, we analyzed allelic transcription for KCNQ1OT1 in these patients and found that 31 (46%) of 67 cases had biallelic expression. Those with biallelic expression all had partial or complete demethylation (MI 0.42), whereas patients with normal monoallelic expression had normal methylation unless UPD for 11p15 was present. These data substantially increase the previous number of cases studied for both KvDRM1 methylation status and KCNQ1OT1 transcription (9). Our data support the contention that there is excellent correlation between KvDMR1 methylation status and allelic transcription of KCNQ1OT1.
We have shown that BWS individuals with constitutional imprinting defects at KvDMR1/KCNQ1OT1 do indeed develop tumors. The tumors observed include hepatoblastoma, rhabdomyosarcoma and gonadoblastoma. What is of particular interest, in our series, is that the rarer types of tumors associated with BWS (rhabdomyosarcoma and gonadoblastoma) are seen in BWS cases with constitutional imprinting defects at KvDMR1/KCNQ1OT1 but usually not in BWS cases with UPD of 11p15 or methylation errors of H19. Moreover, the most common tumor observed in BWS cases, Wilms’ tumor was not seen in patients with KvDMR1/KCNQ1OT1 imprinting errors. This profile of tumor specificity is consistent with available data from molecular analyses of sporadic tumors. For example, imprinting defects of the KCNQ1OT1 transcript or KvDMR1 have not been observed in sporadic Wilms’ tumor (16,30). To date, no data have been reported on the imprinting status of KCNQ1OT1 or KvDMR1 in non-Wilms’ embryonal tumors.
Normal imprinting status of 11p15 in BWS patients
Patients with normal imprinting status of 11p15 (group V) have normal biparental inheritance of 11p15 markers, normal methylation at H19 and KvDMR1 and normal monoallelic expression of KCNQ1OT1. The frequency of tumors for this group in our study is 24%, comparable to 20% in a previous study (25). In addition, all tumors reported to date in this group are Wilms’ tumors. These data, both in terms of frequency and tumor type, are very similar to those seen for BWS molecular defects in the telomeric domain. Given that hemihyperplasia is a frequent finding in group V BWS cases with Wilms’ tumor and that hemihyperplasia is a common finding in BWS cases with 11p15 UPD, we propose that such individuals in group V may represent cryptic cases of 11p15 UPD. Since UPD for 11p15 usually occurs as a somatic mosaic event, one would predict that sampling of multiple tissues from group V BWS patients could potentially demonstrate whether UPD for 11p15 is in fact the underlying molecular defect in at least some group V cases.
Tumor rates in different BWS molecular groups
Children with BWS have a greatly elevated risk of developing a wide variety of pediatric embryonal tumors (1,5,8). Our data show that all four molecular groups are represented in the BWS cases with tumors. We have also reviewed the tumor rates defined for each molecular group from data in the literature. Previous studies that have assessed tumor rates in specific BWS subgroups have concluded that tumors are associated with only two molecular subgroups UPD for 11p15 and H19 hypermethylation. However, such studies have had limited representation of tumors other than Wilms’ tumor and occasionally hepatoblastoma. Two of the largest studies are those of Engel et al. (26) with three Wilms’ tumors and Bliek et al. (25) with seven Wilms’ tumors. Only a recent report by Gaston et al. (29) included a broader tumor spectrum and in this study, one BWS case with an imprinting defect at KvDMR1 was noted to have thyroid carcinoma. Therefore, given that imprinting defects at KCNQ1OT1/KvDMR1 are associated with tumors other than Wilms’ tumor, it is not surprising that investigators have not previously noted the tumor risk for BWS cases with imprinting defects at KCNQ1OT1.
Model for tumor development in BWS
From our data, we propose that specific molecular defects in the telomeric H19 and IGF2 domain have a role in Wilms’ tumorigenesis, whereas the KCNQ1OT1/KvDMR1 centromeric domain is associated with predisposition to other types of embryonal tumors in BWS. Since UPD for 11p15 generally involves both imprinted domains, it is important to define the features which distinguish 11p15 UPD from primary imprinting alterations in the centromeric domain (Fig. 7). In the telomeric domain, it is likely that the critical lesion both for BWS and Wilms’ tumorigenesis is extinction of the H19 gene product via either UPD for 11p15 or methylation of the maternal H19 region (38,39).
We propose that the critical lesion associated with tumor development in the centromeric domain is loss of imprinted gene regulation in cis on the maternally derived chromosome. We expect that this would lead to dysregulation of one or more as yet unidentified downstream targets important for the development of rhabdomyosarcoma, gonadoblastoma and hepatoblastoma. Although CDKNIC is a plausible candidate downstream target, only two tumors, both neuroblastoma, have been reported to date in approximately 38 BWS cases with mutations in CDKNIC (20,21,28,31,41–43). Thus, the key elements in the centromeric domain relevant to non-Wilms’ tumorigenesis remain to be identified. In this regard, other potential downstream targets of KvDMR1 would be excellent candidate genes.
Whereas it is evident that there are at least two genetic pathways leading to tumors in BWS, the finding of biallelic IGF2 expression in some patients with imprinting defects at KCNQ1OT1/KvDMR1 (9) suggests that these pathways may be able to cooperate in tumorigenesis. Loss of imprinting for the IGF2 gene is reported to occur in a large variety of tumors (30). In Wilms’ tumor it may be associated with either H19 hypermethylation or it may occur without any concomitant identified alteration on 11p15. Constitutional loss of imprint of the maternal IGF2 gene has also been reported for sporadic cases of BWS, rarely in association with hypermethylation of the H19 gene, and more commonly without another identified 11p15 defect. Since BWS cases with imprinting defects at KCNQ1OT1/KvDMR1 can show either loss of imprinting or normal imprinting at IGF2 (9), imprinting defects at IGF2 may well be part of the tumorigenic pathways controlled by both the centromeric and telomeric imprinted domains of 11p15.
In summary, we have broadened the scope of epigenetic molecular changes on 11p15 documented for BWS patients who have developed tumors and have defined new associations between specific molecular lesions and the tumor types seen in these patients. These data further elucidate some of the complex genetic pathways leading to BWS tumorigenesis, demonstrating that BWS provides a unique model system to study the networks of interacting genetic pathways subject to constitutional epigenetic alteration that influence tumor risk in children.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Patient material
One hundred and sixty-two patients carrying a diagnosis of BWS were referred for molecular testing. Following review of the clinical features, we confirmed a clinical diagnosis of BWS to 125 patients based on the presence of at least three of the following features: macrosomia, macroglossia, hemihyperplasia, ear creases/pits, abdominal wall defect (omphalocele or umbilical hernia), embryonal tumor; or, at least two of the preceding features plus one of neonatal hypoglycemia, abdominal organomegaly or renal malformation. Eight of the 125 patients had positive family histories. The number of childhood tumors reported in this clinically defined group of BWS cases was 16 (12.8%) and included a broad range of tumor types, specifically Wilms’ tumor, hepatoblastoma, rhabdomyosarcoma and gonadoblastoma.
Control samples were obtained from normal control individuals as well as individuals with balanced chromosome rearrangement not involving chromosome 11.
DNA or RNA was obtained from patient samples for analyses of chromosome 11 UPD, methylation status at H19 and KvDMR, and for KCNQ1OT1 allelic expression. These studies were approved by the Research Ethics Board of the Hospital for Sick Children, Toronto, Canada.
Cell cultures
Lymphoblast lines were maintained in RPM1 1640 media supplemented with 15% fetal calf serum. Fibroblast strains were maintained for fewer than 10 passages in 
-MEM supplemented with 10% fetal calf serum.
Chromosome 11 UPD analysis using quantitative PCR
Genomic DNA was extracted from either peripheral blood, cultured lymphoblasts or skin fibroblast cells from the proband and parents using a QIAamp spin column (QIAGEN) method (as per manufacturer’s instructions). Quantitative multiplex-PCR using highly polymorphic STR markers was performed using three markers within (TH, D11S2362 and D11S1997) and two markers distal (D11S1998 and D11S1974) to the BWS critical region at 11p15.5 in order to detect somatic cell rearrangements giving rise to paternal UPD of the chromosome 11p15 region. Amplification products were separated on a 6% denaturing polyacrylamide gel on an ABI 377 PRISM DNA Sequencer (PE Biosystems, Boston, MA). The allele sizes and corresponding peak areas were determined using Genescan software. The percentage of paternal UPD of alleles at 11p15.5 in the proband was determined from informative alleles at a minimum of two DNA markers within the BWS critical region showing an increase in dosage of >20% based on the following calculation: (Peak area of paternal allele – Peak area of maternal allele) / (Peak area of paternal allele + Peak area of maternal allele).
Analysis of allele-specific KCNQ1OT1 expression
Total RNA was isolated from lymphoblasts or fibroblasts using the TRIZOL® reagent (Gibco BRL, Burlington, Ontario, Canada). mRNA was extracted from total RNA using a QuickPrepTM Micro mRNA purification kit (Amersham Pharmacia Biotech, Little Chalfont, UK). M-MuLV reverse-transcriptase (MBI Fermentas, Burlington, Ontario, Canada) was used for reverse transcription reactions. Screening for contamination by genomic DNA was carried out in an identical tube without reverse transcriptase. RT–PCR products were gel purified and sequenced to determine allele-specific expression.
Southern blot analysis of KvDMR1 and H19 methylation
For analysis of methylation of KvDMR1, genomic DNA was digested with EcoRI and NotI; for analysis of methylation of the H19 gene, genomic DNA was digested with PstI and SmaI. For both methylation assays, digestion products were electrophoresed through 0.8% agarose gels and were then transferred to a GeneScreen Plus membrane (NEN, Boston, MA). For analysis of methylation of KvDMR1, blots were hybridized with the 400 bp [32P]dCTP-labeled DMR probe, a kind gift from M.Higgins (15). For analysis of H19 methylation, blots were hybridized with the H19 promoter probe described by Reik et al. (32). The probes were labeled using the random primed DNA labeling kit (Roche, Mannheim, Germany). The blots were analyzed using a Molecular Dynamics Storm PhosphorImager. The H19 MI was determined by dividing the optical density of the 1.8 kb band by the combined densities of the 1.0 and 1.8 kb bands. For KvDMR1 the MI was determined by dividing the optical density of the 4.2 kb band by the combined densities of the 4.2 and 2.7 kb bands.
CDKNIC mutation screening
The entire coding region of CDKNIC was analyzed by PCR/heteroduplex screening as previously described by Li et al. (31).
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ACKNOWLEDGEMENTS |
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We thank all the families who participated in this study. We would like to acknowledge the expert statistical input of Dr Gideon Koren and the excellent secretarial assistance of Nancy Taylor and Sarah Petty. The KvDMR (DMR) probe was generously provided by Dr M.Higgins. This research was supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society. Ingrid Ambus was the recipient of a Starbucks Clinical Genetics Research Studentship Award.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Division of Clinical and Metabolic Genetics, Hospital for Sick Children, 555 University Avenue,Toronto, Ontario M5G 1X8, Canada. Tel: +1 416 813 6386; Fax: +1 416 813 5345; Email: rweksb@sickkids.on.ca
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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