Human Molecular Genetics, 2001, Vol. 10, No. 5 467-476
© 2001 Oxford University Press
Increased tumour risk for BWS patients correlates with aberrant H19 and not KCNQ1OT1 methylation: occurrence of KCNQ1OT1 hypomethylation in familial cases of BWS
1Department of Clinical Genetics, 2Department of Anatomy and Embryology, 3Department of Pediatrics and 4Institute for Human Genetics, Academic Medical Center, University of Amsterdam, PO Box 22700, 1100 DE Amsterdam, The Netherlands
Received 19 October 2000; Revised and Accepted 5 January 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Beckwith–Wiedemann syndrome (BWS) is an overgrowth malformation syndrome that maps to human chromosome 11p15.5, a region that harbours a number of imprinted genes. We studied the methylation status of H19 and KCNQ1OT1 (LIT1/KvDMR1) in a large series of BWS patients. Different patient groups were identified: group I patients (20%) with uniparental disomy and hence aberrant methylation of H19 and KCNQ1OT1; group II patients (7%) with a BWS imprinting centre 1 (BWSIC1) defect causing aberrant methylation of H19 only; group III patients (55%) with a BWS imprinting centre 2 (BWSIC2) defect causing aberrant methylation of KCNQ1OT1 only; and group IV patients (18%) with normal methylation patterns for both H19 and KCNQ1OT1. BWS patients have an increased risk of developing childhood tumours. In our patient group, out of 31 patients (group III) with KCNQ1OT1 demethylation only, none developed a tumour. However, tumours were found in 33% of patients with H19 hypermethylation (group I and II) and in 20% of patients with no detectable genetic defect (group IV). All four familial cases of BWS showed reduced methylation of KCNQ1OT1, suggesting that in these cases the imprinting switch mechanism is disturbed.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The Beckwith–Wiedemann syndrome (BWS) is a phenotypically heterogeneous overgrowth syndrome that is characterized by a combination of various symptoms. Anterior abdominal wall defects, macroglossia, pre- and postnatal overgrowth and characteristic facial dysmorphology occur in most cases. In addition, neonatal hypoglycaemia, organomegaly, renal abnormalities and hemihypertrophy occur. Children with BWS have an increased susceptibility (7.5%) to childhood tumours, most commonly Wilms’ tumour (WT), but also adrenocortical carcinoma, hepatoblastoma and neuroblastoma (1–7).
Most BWS cases are sporadic but familial inheritance is observed in 15% of all cases. Linkage studies have assigned this syndrome to chromosome 11p15 (8,9). Although most patients have a normal karyotype, a number of chromosomal abnormalities have been described, all involving chromosome 11p15. These abnormalities include maternally inherited balanced chromosomal translocations and inversions that map to three distinct regions on chromosome 11p15: BWS chromosomal regions 1, 2 and 3 (BWSCR1, -2 and -3, respectively) (10–12). Furthermore, paternal trisomy of chromosome 11p15 has been found and in 
20% of the cases uniparental paternal disomies (UPD) of 11p15 could be detected (13–18). In view of the specific parental origin of the chromosomal abnormalities it is evident that imprinting plays a role in the aetiology of the syndrome.
The following genes, located in BWSCR1, are involved in this syndrome (Fig. 1).
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Insulin-like growth factor II (IGF2) is a paternally expressed fetal growth factor. In two studies biallelic expression of this gene was seen in 4/6 (67%) and 9/11 (81%) of the patients without cytogenetic abnormalities (19,20), although in a later study this frequency was only 2/10 patients (20%) (21).
IGF2 has the highest level of expression in tissues that are affected by overgrowth in BWS. Transgenic mice with high over-expression of Igf2 exhibit most BWS symptoms, except tumours, hemihypertrophy, hypoglycaemia and exomphalos (22).
H19 is a maternally expressed gene that codes for an untranslated RNA. Its mode of action is not yet fully understood. H19 and IGF2 are reciprocally imprinted. Deletion of H19 leads to loss-of-imprinting (LOI) and biallelic expression of Igf2 in the mouse. These mice have an increased birth weight (23–25). The mechanism through which Igf2 is controlled by H19 is being debated: H19 functions according to either an enhancer competition model or a more recently proposed boundary model (26–28). Regulation of IGF2 expression is the most probable function of the gene.
H19 is expressed in human fetal tissues and down-regulated postnatally but re-expressed in tumours arising from tissues that express the gene during embryogenesis (29,30).
LOI of H19 is observed in 8% of sporadic non-UPD BWS patients, leading to inactivation of the maternal H19 gene and activation of the maternal IGF2 gene (31).
CDKNIC(p57kip2) encodes a cyclin-dependent kinase (Cdk) inhibitor, involved in the regulation of the cell cycle. CDKNIC is expressed from the maternal allele only, although some expression (5–30%) is observed from the paternal chromosome (32). The first study of CDKNIC in BWS patients reported a high percentage (20%) of mutations in this gene (33). In more recent studies, however, a lower percentage (<10%) was found in sporadic cases. Of familial cases 40% harboured CDKNIC mutations (34–38). Mice lacking p57kip2 expression display some of the BWS symptoms like exomphalos and adrenal cortex dysplasia but lack features typical for BWS like gigantism and macroglossia (39).
BWS patients with UPD of IGF2 and those with mutations in CDKNIC are phenotypically indistinguishable. It was suggested that CDKNIC and IGF2 act in opposing ways to control cell cycle proliferation during development. A double mutant mouse model, in which both loss-of-function of p57kip2 and LOI of Igf2 are present, showed indeed that CDKNIC and IGF2 do interact (40).
KCNQ1(KvLQT1) codes for a voltage-gated potassium channel. The gene spans 300 kb and encompasses all translocation breakpoints in BWSCR1 (10,41). KCNQ1 is expressed only from the maternal allele except in the heart where both copies of the gene are expressed (42). In two patients with a chromosomal translocation disrupting KCNQ1, biallelic expression of IGF2 was found (43,44) but the exact role of KCNQ1 in the aetiology of BWS is not yet understood.
Recently a fifth gene in BWSCR1 involved in the development of BWS, KCNQ1OT1 (KCNQ1 overlapping transcript 1), was discovered. Mitsuya et al. (45) described the existence of a long QT intronic transcript 1 (LIT1) within KCNQ1 that was transcribed in the antisense orientation. KCNQ1OT1 codes for a very large (>80 kb) RNA that is expressed only from the paternal allele. Lee et al. (21) demonstrated that 8/16 (50%) BWS patients show biallelic expression of KCNQ1OT1 whereas 21/36 (58%) patients showed complete loss of maternal allele-specific methylation of a CpG island upstream of KCNQ1OT1. LOI of KCNQ1OT1 was not linked to LOI of IGF2 (21).
This CpG island has also been described by Smilinich et al. (46) and named potassium voltage differentially methylated region 1 (KvDMR1). The antisense RNA associated with this CpG island was called KvLQT1-AS. Demethylation of KvDMR1 was seen in 5/12 BWS cases with normal H19 methylation, and in 4/4 cases with H19 hypermethylation the methylation pattern of KvDMR1 was conserved. Demethylation of KCNQ1OT1 is thus the most common genetic alteration in BWS.
This study describes the methylation patterns of both KCNQ1OT1 and H19 in a large series of BWS patients. Clinical data were collected from all patients to ascertain the BWS diagnosis and to allow detection of a possible correlation between tumour risk and genotype. All patients were screened for CDKNIC mutations.
Our results suggest that tumour risk is not associated with demethylation of KCNQ1OT1 but correlates with hypermethylation of H19. In contrast to findings of Lee et al. (21) and Smilinich et al. (46), partial demethylation of KCNQ1OT1 (LIT1) was seen in a large proportion of non-UPD BWS patients. In UPD patients, hypomethylation of KCNQ1OT1 correlates with the fraction of uniparental cells, as does the hypermethylation of H19. In all familial cases of BWS, hypomethylation of KCNQ1OT1 was found. In our population the incidence of CDKNIC mutations is low (<1%).
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Patient material
In total, 115 patients who had been diagnosed with BWS were referred to our hospital for routine diagnostics. Clinical data were collected from 101 patients and, according to the data, the diagnosis BWS was re-examined (Table 1). Twenty-eight patients were classified EE when they met the very strict criteria of Elliot (5), i.e. three major features or two major features plus three or more minor features (major features, anterior abdominal wall defects, macroglossia and pre- and/or postnatal growth >90th centile; minor features, ear creases or pits, naevus flammeus, hypoglycaemia, nephromegaly and hemihypertrophy). Forty-five patients were classified ED when they met the less strict criteria of DeBaun (7), i.e. two or more of the five most common features (macroglossia, birth weight >90th percentile, hypoglycaemia in the first month of life, ear creases/pits and abdominal wall defects). Six patients had some of the clinical features of BWS but did not meet either the Elliot or the DeBaun criteria and were classified as D (doubtful). An additional 16 patients did not meet any of the major criteria and were classified as being non-BWS (N). No additional clinical data were available for three patients (T) who developed a Wilms’ tumour. Three patients (Chrom.) had a chromosomal abnormality—a translocation on chromosome 11p15.5; no clinical data were available for the remaining 14 patients (X). The series includes four familial cases: unrelated patients of which one or more first-degree relatives show signs of BWS. In detail, the mother of B61 presented with ear creases whilst the brother of this patient presented with ear creases and macroglossia. The mother of B84 presented with macroglossia at birth. B89 had a sister with macroglossia and the grandmother of B135 had an umbilical hernia, macroglossia and ear creases whilst his father had macroglossia (>90th centile at birth).
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CDKNIC mutations
PCR followed by SSCP analysis was performed to screen 102 patients for mutations in CDKNIC. We detected a W50S mutation in only one patient (1%), which was inherited from the mother. The causal relation of this mutation and the BWS phenotype is unclear.
Uniparental disomies
For the detection of uniparental disomies we screened a series of polymorphic markers on 11p15. In a series of 113 patients, we detected 17 cases (15%) in which the cells had become disomic for the paternal chromosome (Table 1). These uniparental disomies were always present in a mosaic form, with the increase in paternal contribution varying from 37 to 90%. The smallest region of disomy ranges from D11S860 to D11S2071 (M. Alders, unpublished data).
The overall risk in BWS patients for the development of childhood tumours in our population is 7.1% (8/113), which is in agreement with the 7.5% found in the literature (1–7). Of the 17 UPD cases, three patients developed a Wilms’ tumour and one patient developed a hepatoblastoma, in total 4/17 (24%).
Of the non-UPD cases, four patients developed a Wilms’ tumour, 4/96 (4%). Although the highest risk for the development of tumours is found in the UPD group, the non-UPD group still has an increased risk for malignancies in comparison with the general population.
Methylation status of H19
We analysed the methylation status of the differentially methylated SmaI site near the promoter of H19 by Southern blot analysis, as described by Reik et al. (47). To monitor complete digestion by SmaI we used an 800 bp fragment (SmaC) localized on chromosome 1p34.1–p35 as a control probe. This probe recognizes a non-methylated SmaI site (Fig. 2A).
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For each patient we calculated a methylation index (M.I.) as follows. The mean M.I. of a control group was 0.50 with a standard deviation (SD) of 0.03 (n = 22). Therefore, we set the borderline for hypermethylation at M.I.H19
0.56 (mean + 2 x SD). In 19 out of 102 (19%) patients we found hypermethylation of H19 (Table 1). If we limit the patient group to only those patients with confirmed clinical diagnosis, 16/64 (25%) show hypermethylation of H19. This group includes all UPD cases of which H19 data were available.
Childhood tumours were found in 5/19 (26%) of the patients with hypermethylation of H19 (two non-UPD and three UPD cases). All four familial BWS cases showed a normal methylation pattern for H19.
Methylation status of KCNQ1OT1
To examine the methylation status of KCNQ1OT1 we analysed a differentially methylated NotI site in the CpG island upstream of KCNQ1OT1 by Southern blotting. To monitor complete digestion by NotI we used an 800 bp fragment located on chromosome 1p36 (NotC) as a control probe (Fig. 2B). The M.I. was calculated in the same way as for H19.
In a control group we obtained a mean M.I. of 0.51 with an SD of 0.025 (n = 20), therefore we set the borderline for hypomethylation of KCNQ1OT1 at M.I.KCNQ1OT1 
0.45 (mean – 2 x SD).
Fifty out of the 89 (64%) patients analysed had a decreased M.I.KCNQ1OT1 (Table 1). If we limit the group to only those patients with confirmed clinical diagnosis, the percentage is even higher, 41/55 (75%). It is remarkable that of the patients with only minor clinical BWS features (Table 1, N and D), 4/19 display demethylation of KCNQ1OT1. The clinical data of these four patients are listed in Table 2. These patients do not comply with either the EE or ED criteria.
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The degree of demethylation of KCNQ1OT1 varies from 10 to 100%. As would be expected, all UPD cases of which KCNQ1OT1 data are available had a decreased M.I.KCNQ1OT1. Of all the non-UPD patients with hypermethylation of H19, the M.I. of KCNQ1OT1 was normal, supporting the fact that these patients indeed do not have an imbalance in parental allelic contribution.
Two out of 50 (4%) of the patients with a decreased M.I.KCNQ1OT1 developed a childhood tumour; both patients displayed UPD.
But the most striking observation was that there were no tumours found in the non-UPD cases with hypomethylation of KCNQ1OT, reducing the tumour risk of this group to 0%. All four familial BWS cases show reduced methylation of KCNQ1OT1.
Combined methylation analysis of KCNQ1OT1 and H19
We were able to perform both KCNQ1OT1 and H19 analysis in 56 patients. Based on the molecular data for these two genes, four different patient groups could be distinguished. A summary of the results is listed in Table 3.
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Group I: UPD resulting in an M.I.KCNQ1OT1 0.45 and M.I.H19 0.56. Eleven out of 56 (20%) patients had an M.I.H19 0 .56 and M.I.KCNQ1OT1 0.45 (Table 4). These were all UPD cases; the aberrant methylation pattern in this group was due to the presence of extra paternal material.
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Group II: methylation error of H19, resulting in an M.I.KCNQ1OT1 = 0.50 and M.I.H19 0.56. Four out of 56 (7%) patients had an M.I.H19 0.56 but an M.I.KCNQ1OT1 = 0.50 (Table 5). All four patients in this group had no obvious evidence of UPD. Of these patients, 2/4 (50%) developed a Wilms’ tumour.
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Group III: methylation error of KCNQ1OT1, resulting in an M.I.H19 = 0.50 and M.I.KCNQ1OT1 0.45. Thirty-one out of 56 (55%) of the BWS patients display normal H19 methylation and hypomethylation of KCNQ1OT1 (Table 6). None of these patients developed a tumour. In this group all four familial BWS cases are included, all displaying hypomethylation of KCNQ1OT1 and normal H19 methylation.
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Group IV: normal methylation pattern. Ten out of 56 (18%) of the BWS patients display normal methylation of H19 and also normal methylation of KCNQ1OT1 (Table 7). Of these patients, two (20%) developed a Wilms’ tumour. Both patients demonstrate classical features of BWS (weight at birth > P97, hemihypertrophy, macroglossia and nephromegaly). The patient with the CDKNIC mutation falls into this group, displaying normal methylation for both KCNQ1OT1 and H19.
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Statistical analysis
UPD. To test the reliability of the method used to detect methylation defects we compared the percentage of uniparental cells with the methylation indices of KCNQ1OT1 and H19. In UPD patients the sum of M.I.KCNQ1OT1 and M.I.H19 is expected to equal 1, where M.I.H19 is expected to equal the fraction of UPD cells and M.I.KCNQ1OT1 is expected to equal (1 – the fraction of disomic cells).
The methylation indices of KCNQ1OT1 and H19 do not differ significantly from the fraction of uniparental cells: t(1 – UPD + KCNQ1OT1) = 0.459 (P = 0.657) and t(UPD + H19) = 1.666 (P = 0.130). The sum of M.I.KCNQ1OT1 and M.I.H19 does not differ from 1 (t = 1.761; P = 0.109)
The high P-value of the comparison of (1 – the fraction of disomic cells) and M.I.KCNQ1OT1 indicates that the method to measure the M.I. of KCNQ1OT1 is an accurate alternative for the standard method to calculate the fraction UPD cells.
Tumour risk. The tumour incidence for patients with aberrant H19 methylation (5/15) is significantly different from patients with a normal H19 methylation pattern (2/41): Z = 2.85; P = 0.004. The difference in tumour incidence for patients with (3/42) and without (4/14) aberrant KCNQ1OT1 methylation is less significant: Z = 2.10; P = 0.036.
Tumour incidence in the four groups (Table 3) was statistically different (
2 = 12.3; P = 0.006) but groups I, II and IV were similar (2 = 1.28; P = 0.527) and could therefore be pooled for further analysis. A comparison of tumour incidence of group III (0/31) and groups I, II and IV (7/25) showed a significant difference (Z = 3.15; P = 0.002). The number of cases in this test is such that even when one tumour had occurred in group III the difference in tumour incidence would still have been identified as significant (at significance level of 0.01) with a power of 0.90.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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At the molecular level (methylation studies) at least four patient categories can be identified. In addition, familial cases with aberrant methylation of KCNQ1OT1 were found. Mutations in CDKNIC were only found in a single patient of Asian origin.
Imbalance in parental allelic contribution
Group I patients (20% of the patients) are all UPD cases. This group has a high risk of developing childhood neoplasias (27%). The increased paternal contribution of the genome results in hypermethylation of H19 and hypomethylation of KCNQ1OT1. We found 15% uniparental disomy cases in our patient population, which is consistent with other studies (1–6). Because of the imbalance in parental contribution to the genome of these patients, the methylation pattern of genes in the region will show the same imbalance. Since all UPD cases are of paternal origin, the excess of paternal contribution to the genome will result in a shift towards the paternal methylation status of all imprinted genes in the disomic region.
In our series, all UPD cases are mosaic and show only partial demethylation of KCNQ1OT1 and hypermethylation of H19, concordant with the expected character of UPD. UPD fraction and methylation indices of both genes do not differ. Aberrant methylation of both KCNQ1OT1 and H19 in group I patients is therefore a direct result of the over-representation of the paternal genome, rather than a result of an imprinting defect such as mutation of an imprinting centre (IC).
In group I the M.I. of KCNQ1OT1 plus the M.I. of H19 do not differ from 1. This indicates that the method used to measure the methylation indices is very accurate.
The smallest region of disomy (SRD) spans a large region on chromosome 11p15, at least from D11S2071 to D11S860 (unpublished data). The region encompasses all genes in BWSCR1 known to be involved in the BWS phenotype (CDKNIC, KCNQ1OT1, IGF2 and H19) but also other imprinted genes (TAPA1, TSSC3 and TSSC5) that may play a role in the aetiology of the syndrome.
All patients in this group I meet the criteria of either Elliot or DeBaun and, hence, are very obvious BWS cases. Moreover, they have a high risk of developing childhood tumours. Therefore the region must contain at least one gene involved in the overgrowth phenotype and at least one gene involved in tumour development. IGF2 remains a strong candidate for both functions. Overexpression of Igf2 in mice results in an overgrowth phenotype resembling the BWS phenotype. Overexpression and LOI of IGF2 is reported in many tumour types (48). Transgenic mice overexpressing Igf2 with tissue-specific and temporally controlled promoters develop tumours. However, there is a latent period of at least a few months, indicating that additional events must occur before tumours arise (49,50). Such an additional event might be the inactivation of a tumour suppressor gene located in the disomic region.
Methylation defects of H19
In group II patients (7% of all patients), hypermethylation of H19 is not due to an imbalance in parental contribution because these patients do not show UPD. The patients have a normal M.I. of KCNQ1OT1, limiting the region of aberrant methylation to the region containing H19.
The increase in the M.I. of H19 must be caused by a mechanism that results in methylation of the normally unmethylated maternal copy of H19. The postulated BWSIC1 is thought to control the methylation of genes in this region. (51). The localization and the exact mechanism through which this centre exhibits its function are unknown but candidate regions in the mouse are a CpG site immediately upstream of H19 and the intergenic region between H19 and Igf2 (26).
Reik et al. (52) demonstrated in fibroblasts the alteration of the imprinting pattern of IGF2 for patients with hypermethylation of H19. It is therefore likely that the imprinting control mechanism extends its action over a larger region surrounding H19, including IGF2 but excluding KCNQ1OT1.
Methylation defects of KCNQ1OT1
In group III patients (55% of all patients), demethylation of KCNQ1OT1 must be caused by disruption of the mechanism that regulates the methylation of the maternal copy of KCNQ1OT1. In this group the normally methylated maternal copy of the gene is demethylated. KCNQ1OT1 methylation is under the control of a postulated BWSIC2, which might also control IGF2 and possibly CDKNIC imprinting (51). Lee et al., however, demonstrated that UPD of KCNQ1OT1 is independent of IGF2 imprinting (21), excluding IGF2 from the region that is controlled by BWSIC2.
In a previous study, Lee et al. (21) demonstrated that for all patients that show LOI of KCNQ1OT1, demethylation of the maternal allele was always complete. They do not describe, however, whether their series includes any UPD patients. Smilinich et al. (46) analysed both UPD and non-UPD cases, and of six non-UPD cases, three showed complete demethylation of KCNQ1OT1 whereas the other three had a normal methylation pattern. This does not correspond to our findings: out of 40 non-UPD patients with KCNQ1OT1 demethylation, only 11 show complete loss of methylation of the maternal allele; all others show partial demethylation. The high frequency of KCNQ1OT1 methylation defects without H19 methylation defects seems to contrast with the high frequency of aberrant IGF2 expression published by others. However, we have not studied the IGF2 expression directly.
Tumour risk is associated with H19 methylation defects and not with KCNQ1OT1 methylation defects
The difference in tumour incidence between group III, patients with aberrant methylation of KCNQ1OT1 and normal methylation of H19, and the other groups, clearly indicates that the tumour risk in group III is significantly lower then in the other groups. Tumour risk is therefore not associated with demethylation of KCNQ1OT1.
However, the tumour incidence for patients with an increased M.I. of H19 (group I and II patients) is increased compared with patients with a normal M.I. of H19. Hypermethylation of H19 is therefore positively associated with tumour risk.
This indicates that the postulated tumour suppressor gene must be located in the region associated with only group I and II patients, i.e. BWSIC1.
Dao et al. have already demonstrated that, in Wilms’ tumours, silencing by methylation is restricted to H19 (53). They also analysed other genes in the region (IPL, IMPT1, ZNF195 and KCNQ1OT1) but found no changes in methylation patterns in these genes. H19 was also hypermethylated in pre-neoplastic WT-associated kidneys where KCNQ1OT1 demethylation was not observed.
Familial BWS cases
All four familial cases in our series show demethylation of KCNQ1OT1 in the absence of a methylation defect at the H19 locus. In these familial cases the transmitted genetic defect must be the inability to establish or maintain the maternal methylation pattern of KCNQ1OT1 on the maternal chromosome.
In the families described in the literature, BWS is predominantly transmitted through females (54–57). Moutou et al. (56) described a lower penetrance and a milder phenotype for paternal transmittance of BWS. Viljoen and Ramesar (57) collated 27 kindred from the literature and in most families the mode of inheritance is sex related, i.e. the disease is transmitted through carrier males and females, but only in the offspring of female carriers do as many as 50% display the phenotype.
Analogous to Angelman syndrome (AS), this might be due to the disruption of the paternal-to-maternal imprint switch (58–62) (Fig. 3). Prader–Willi syndrome (PWS) and AS result from molecular defects in chromosome region 15q11–q13 that cause loss of expression of paternally or maternally transcribed genes, respectively. In some PWS or AS families, small deletions or mutations in the ICs in this region prevent imprinting switching. Mutations in the AS-IC in a male ancestor prevent resetting of the paternal imprint in his (unaffected) daughters, causing AS in 50% of his grandchildren. Although in our cases the grandparental origin of the paternally imprinted maternal chromosome is not known, a similar mechanism could be involved.
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BWS patients without methylation defects in H19 and KCNQ1OT1
In group IV patients (18% of all patients) no methylation defects in either KCNQ1OT1 or H19 were found. In view of the lack of reciprocity of H19 and IGF2 imprinting found by Joyce et al. (20), it is possible that IGF2 is activated in an H19-independent pathway. H19 silencing leads to IGF2 activation through either the enhancer-competition mechanism or the chromatin boundary model. Through this mechanism, overexpression of IGF2 would be causative for the BWS phenotype in this group of patients.
Furthermore, it was demonstrated by Smilinich et al. (46) that patients with CDKNIC mutations show no aberrant methylation of KCNQ1OT1 or H19. The only patient in our series who displays a CDKNIC mutation does indeed have normal methylation patterns for both genes. Through the proposed interaction between CDKNIC and Igf2 in the mouse (40) mutations in CDKNIC could also lead to over-expression of IGF2, and hence the BWS phenotype.
Two patients in group IV developed a Wilms’ tumour, indicating that either of the proposed mechanisms can still include the inactivation of a tumour suppressor gene.
In summary, we describe the analysis of BWS-associated molecular defects in a large series of patients. In the majority of BWS patients, methylation defects of KCNQ1OT1 and/or H19 were found. These include familial cases. In contrast with other studies, this hypo/hypermethylation of KCNQ1OT1 was often not complete. Whenever UPD was seen, the change in methylation patterns correlated with the UPD. Tumours were seen only in patients with H19 hypermethylation or patients with no obvious genetic defects. No tumours were seen in patients with hypomethylation of KCNQ1OT1 only. Although the data strongly suggest that this latter category of patients has no increased tumour risk, more studies will be needed before a negative screening advice can be given.
This study shows that methylation studies considerably improve the diagnostics for BWS patients compared with cytogenetic and UPD screening that have been used as diagnostic tools thus far. However, care should be taken that hypomethylation of KCNQ1OT1 was also found in patients demonstrating only part of the classical BWS phenotype. Therefore, this test might be applicable to a much larger group of patients.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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CDKNIC mutation analysis
DNA was extracted from peripheral blood lymphocytes by using standard protocols. The complete cDNA sequence of the gene, except the PAPA repeat, was amplified in overlapping PCR fragments (primers listed in Table 8). PCR products were subsequently separated on a 12.5% non-denaturing polyacrylamide gel (Pharmacia Biotech) at 5 and 15°C and stained using the Silver Staining kit (Pharmacia Biotech). PCR fragments revealing an aberrant separation pattern were re-amplified from DNA and sequenced by the fluorescent dideoxy chain termination method on an ABI 377 (Applied Biosystems).
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UPD screening
The following probes were used for Southern blot analysis: p2.1 (37), pEJ6.6 (HRAS) (38) and pHd3.2 (HBBC) (39). The CA-repeat markers D11S1318, D11S988 and TH were amplified from DNA by PCR as described in the Genome Database (http:\\www.gdb.org) with the use of Cy5 end-labelled primers. The fluorescence intensity of PCR products was subsequently measured on the ALFexpress electrophoresis system and analysed using ALF Fragment Manager software (Pharmacia Biotech).
The percentage disomic cells was calculated as follows:
%UPD = [(p – m)/(p + m)] x 100%
(p, intensity paternal allele; m, intensity maternal allele).
Methylation screening
For Southern blot analysis, 11 µg of genomic DNA was first digested overnight with a methylation-sensitive restriction enzyme (NotI for KCNQ1OT1 and SmaI for H19), precipitated and digested overnight with a second restriction enzyme (BamHI for KCNQ1OT1 and PstI for H19). DNA was separated on an agarose gel and transferred to a Hybond N+ membrane (Amersham). The membrane was subsequently hybridized with [
-32P]dCTP-labelled fragment using the random primed method. Hybridization was performed overnight in ExpresseHyb (Clontech) and the filters were washed in 2x SSC/0.1% SDS prior to exposure in a phosphorimager (Amersham). The imagequant program (Amersham) was used to measure the intensity of the radioactive bands.
Probes were made by PCR (primers listed in Table 8). Completion of digestion of the methylation-sensitive enzyme was monitored by the use of a control probe (NotC for KCNQ1OT1 and SmaC for H19) that recognizes a non-methylated restriction site.
Statistical analysis
t-tests were used to test the null hypothesis that M.I.KCNQ1OT1 + M.I.H19 = 1 (one-sample test) and that M.I.H19 = the UPD fraction and M.I.KCNQ1OT1 = 1 – the UPD fraction (paired tests).
Tumour incidences in four patient groups, based on the normal and/or aberrant methylation of KCNQ1OT1 and H19, were compared with a 2 test. Since groups I, II and IV showed similar incidences, these groups were pooled and the tumour incidence in this pooled group was compared with group III with a Z-test based on the normal approximation of the binomial distribution (63)
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ACKNOWLEDGEMENTS |
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We would like to thank Karin Kroeze-Jansema, Karin van der Lip and Sebastiaan de Leng for excellent technical assistance and the physicians who referred their patients to our clinic. We are grateful for the willingness of the patients and their parents to contribute to this study, in particular Mr L. van Oostende of the Dutch BWS Patients Association. This work was supported by the Netherlands Organisation of Scientific Research (NWO grant 901-04-208).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +31 205667218; Fax: +31 206918626; Email: m.a.mannens@amc.uva.nl
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REFERENCES |
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