New chromosome 11p15 epigenotypes identified in male monozygotic twins with Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome demonstrating heterogeneous molecular alterations of two imprinted domains on chromosome 11p15. The most common molecular alterations include loss of methylation at the proximal imprinting center, IC2, paternal uniparental disomy (UPD) of chromosome 11p15 and hypermethylation at the distal imprinting center, IC1. An increased incidence of female monozygotic twins discordant for BWS has been reported. The molecular basis for eleven such female twin pairs has been demonstrated to be a loss of methylation at IC2, whereas only one male monozygotic twin pair has been reported with this molecular defect. We report here two new pairs of male monozygotic twins. One pair is discordant for BWS; the affected twin exhibits paternal UPD for chromosome 11p15 whereas the unaffected twin does not. The second male twin pair is concordant for BWS and both twins of the pair demonstrate hypermethylation at IC1. Thus, this report expands the known molecular etiologies for BWS twins. Interestingly, these findings demonstrate a new epigenotype-phenotype correlation in BWS twins. That is, while female monozygotic twins with BWS are likely to show loss of imprinting at IC2, male monozygotic twins with BWS reflect the molecular heterogeneity seen in BWS singletons. These data underscore the need for molecular testing in BWS twins, especially in view of the known differences among 11p15 epigenotypes with respect to tumor risk.     

Epigenetic alterations of H19 and LIT1 distinguish patients with Beckwith-Wiedemann syndrome with cancer and birth defects

Beckwith-Wiedemann syndrome (BWS) is a congenital cancer-predisposition syndrome associated with embryonal cancers, macroglossia, macrosomia, ear pits or ear creases, and midline abdominal-wall defects. The most common constitutional abnormalities in BWS are epigenetic, involving abnormal methylation of either H19 or LIT1, which encode untranslated RNAs on 11p15. We hypothesized that different epigenetic alterations would be associated with specific phenotypes in BWS. To test this hypothesis, we performed a case-cohort study, using the BWS Registry. The cohort consisted of 92 patients with BWS and molecular analysis of both H19 and LIT1, and these patients showed the same frequency of clinical phenotypes as those patients in the Registry from whom biological samples were not available. The frequency of altered DNA methylation of H19 in patients with cancer was significantly higher, 56% (9/16), than the frequency in patients without cancer, 17% (13/76; P=.002), and cancer was not associated with LIT1 alterations. Furthermore, the frequency of altered DNA methylation of LIT1 in patients with midline abdominal-wall defects and macrosomia was significantly higher, 65% (41/63) and 60% (46/77), respectively, than in patients without such defects, 34% (10/29) and 18% (2/11), respectively (P=.012 and P=.02, respectively). Additionally, paternal uniparental disomy (UPD) of 11p15 was associated with hemihypertrophy (P=.003), cancer (P=.03), and hypoglycemia (P=.05). These results define an epigenotype-phenotype relationship in BWS, in which aberrant methylation of H19 and LIT1 and UPD are strongly associated with cancer risk and specific birth defects.     

Beckwith–Wiedemann syndrome prenatal diagnosis by methylation analysis in chorionic villi

Beckwith–Wiedemann syndrome (BWS) is an imprinting disorder that can be prenatally suspected or diagnosed based on established clinical guidelines. Molecular confirmation is commonly performed on amniocytes. The possibility to use fresh (CVF) and cultured (CVC) chorionic villi has never been investigated. To verify whether CVF and CVC are reliable sources of DNA to study fetal methylation, we used pyrosequencing to test the methylation level of a number of differentially methylated regions (DMRs) at several imprinted loci (ICR1, ICR2, H19, PWS/AS-ICR, GNASXL, GNAS1A, ZAC/PLAGL1, and MEST) and at non-imprinted MGMT and RASSF1A promoters. We analyzed these regions in 19 healthy pregnancies and highlighted stable methylation levels between CVF and CVC at ICR1, ICR2, GNASXL, PWS/AS-ICR, and MEST. Conversely, the methylation levels at H19 promoter, GNAS1A and ZAC/PLAGL1 were different in CVC compared to fresh CV. We also investigated ICR1 and ICR2 methylation level of CVF/CVC of 2 BWS-suspected fetuses (P1 and P2). P1 showed ICR2 hypomethylation, P2 showed normal methylation at both ICR1 and ICR2. Our findings, although limited to one case of BWS fetus with an imprinting defect, can suggest that ICR1 and ICR2, but not H19, could be reliable targets for prenatal BWS diagnosis by methylation test in CVF and CVC. In addition, PWS/AS-ICR, GNASXL, and MEST, but not GNAS1A and ZAC/PLAGL1, are steadily hemimethylated in CV from healthy pregnancies, independently from culture. Thus, prenatal investigation of genomic imprinting in CV needs to be validated in a locus-specific manner.     

High frequency of copy number variations (CNVs) in the chromosome 11p15 region in patients with Beckwith–Wiedemann syndrome

Beckwith–Wiedemann syndrome (BWS), an overgrowth and tumor predisposition syndrome is clinically heterogeneous. Its variable presentation makes molecular diagnosis particularly important for appropriate counseling of patients with respect to embyronal tumor risk and recurrence risk. BWS is characterized by macrosomia, omphalocele, and macroglossia. Additional clinical features can include hemihyperplasia, embryonal tumors, umbilical hernia, and ear anomalies. BWS is etiologically heterogeneous arising from dysregulation of one or both of the chromosome 11p15.5 imprinting centers (IC) and/or imprinted growth regulatory genes on chromosome 11p15.5. Most BWS cases are sporadic and result from loss of maternal methylation at imprinting center 2 (IC2), gain of maternal methylation at imprinting center 1 (IC1) or paternal uniparental disomy (UPD). Heritable forms of BWS (15 %) have been attributed mainly to mutations in the growth suppressor gene CDKN1C, but have also infrequently been identified in patients with copy number variations (CNVs) in the chromosome 11p15.5 region. Four hundred and thirty-four unrelated BWS patients referred to the molecular diagnostic laboratory were tested by methylation-specific multiplex ligation-dependent probe amplification. Molecular alterations were detected in 167 patients, where 103 (62 %) showed loss of methylation at IC2, 23 (14 %) had gain of methylation at IC1, and 41 (25 %) showed changes at both ICs usually associated with paternal UPD. In each of the three groups, we identified patients in whom the abnormalities in the chromosome 11p15.5 region were due to CNVs. Surprisingly, 14 patients (9 %) demonstrated either deletions or duplications of the BWS critical region that were confirmed using comparative genomic hybridization array analysis. The majority of these CNVs were associated with a methylation change at IC1. Our results suggest that CNVs in the 11p15.5 region contribute significantly to the etiology of BWS. We highlight the importance of performing deletion/duplication testing in addition to methylation analysis in the molecular investigation of BWS to improve our understanding of the molecular basis of this disorder, and to provide accurate genetic counseling.     

Alterations of H19 imprinting and IGF2 replication timing are infrequent in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder resulting from dysregulation of multiple imprinted genes through a variety of distinct mechanisms. A frequent alteration in BWS involves changes in the imprinting status of the coordinately regulated IGF2 and H19 genes on 11p15. Patients have been categorized according to alterations in the imprinted expression, allele-specific methylation, and regional replication timing of these genes. In this work, IGF2/H19 expression, H19 DNA methylation, and IGF2 regional replication timing were studied in nine karyotypically normal BWS fibroblasts and two BWS patients with maternally inherited 11p15 chromosomal rearrangements. Informative patients (9/9) maintained normal monoallelic H19 expression/methylation, despite biallelic IGF2 expression in 6/9. Replication timing studies revealed no changes in the pattern of asynchronous replication timing for both a patient with biallelic IGF2 expression and a patient carrying an 11p15 inversion. In contrast, a patient with a chromosome 11;22 translocation and normal H19 expression/methylation exhibited partial loss of asynchrony and a shift toward earlier replication times. These results indicate that in BWS, (1) H19 imprinting alterations are less frequent than previously estimated, (2) IGF2 imprinting and H19 imprinting are not necessarily coordinated, and (3) alterations in regional replication timing are generally not correlated with either chromosomal rearrangements or the imprinting status of IGF2 and H19.     

Epigenetic deregulation of imprinting in congenital diseases of aberrant growth

Human chromosome 11p15 comprises two imprinted domains important in the control of fetal and postnatal growth. Novel studies establish that imprinting at one of these, the IGF2-H19 domain, is epigenetically deregulated (with loss of DNA methylation) in Silver-Russell Syndrome (SRS), a congenital disease of growth retardation and asymmetry. Previously, the exact opposite epigenetic alteration (gain of DNA methylation) had been detected at the domain's 'imprinting control region' (ICR) in patients with Beckwith-Wiedemann Syndrome (BWS), a complex disorder of fetal overgrowth. However, more frequently, BWS is caused by loss of DNA methylation at the ICR that regulates the second imprinted domain at 11p15. Interestingly, a similar epigenetic alteration (with loss of methylation) at a putative ICR on human chromosome 6q24, is involved in transient neonatal diabetes mellitus (TNDM), a congenital disease with intrauterine growth retardation and a transient lack of insulin. Thus, fetal and postnatal growth is epigenetically controlled by different ICRs, at 11p15 and other chromosomal regions.     

Mitotic recombination and uniparental disomy in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is a model human imprinting disorder resulting from altered activity of one or more genes in the 11p15.5 imprinted gene cluster. Approximately 20% of BWS cases have uniparental disomy (UPD) of chromosome 11. Such cases appear to result from mitotic recombination occurring in early embryogenesis and offer a rare opportunity to study mitotic recombination in nonneoplastic cells. We analyzed a cohort of 52 children with BWS and UPD using a panel of microsatellite markers for chromosome 11. All cases demonstrated mosaic paternal isodisomy, and IGF2 and H19 were included in the segment of UPD in all cases. However, the extent of segmental disomy was variable, with no evidence of clustering of the proximal UPD breakpoint. In most cases (92% of those informative) UPD did not involve 11q, but 4 patients demonstrated UPD for the whole of chromosome 11. In contrast to meiotic recombination, the mitotic recombination frequency did not decline near the centromere.     

Maternal gametic transmission of translocations or inversions of human chromosome 11p15.5 results in regional DNA hypermethylation and downregulation of CDKN1C expression

Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome associated with genetic or epigenetic alterations in one of two imprinted domains on chromosome 11p15.5. Rarely, chromosomal translocations or inversions of chromosome 11p15.5 are associated with BWS but the molecular pathophysiology in such cases is not understood. In our series of 3 translocation and 2 inversion patients with BWS, the chromosome 11p15.5 breakpoints map within the centromeric imprinted domain, 2. We hypothesized that either microdeletions/microduplications adjacent to the breakpoints could disrupt genomic sequences important for imprinted gene regulation. An alternate hypothesis was that epigenetic alterations of as yet unknown regulatory DNA sequences, result in the BWS phenotype. A high resolution Nimblegen custom microarray was designed representing all non-repetitive sequences in the telomeric 33 Mb of the short arm of human chromosome 11. For the BWS-associated chromosome 11p15.5 translocations and inversions, we found no evidence of microdeletions/microduplications. DNA methylation was also tested on this microarray using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. This high-resolution DNA methylation microarray analysis revealed a gain of DNA methylation in the translocation/inversion patients affecting the p-ter segment of chromosome 11p15, including both imprinted domains. BWS patients that inherited a maternal translocation or inversion also demonstrated reduced expression of the growth suppressing imprinted gene, CDKN1C in Domain 2. In summary, our data demonstrate that translocations and inversions involving imprinted domain 2 on chromosome 11p15.5, alter regional DNA methylation patterns and imprinted gene expression in cis, suggesting that these epigenetic alterations are generated by an alteration in "chromatin context".     

Novel mutations of CDKN1C in Japanese patients with Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an imprinting-related human disease that is characterized by macrosomia, macroglossia, abdominal wall defects, and variable minor features. BWS is caused by several genetic/epigenetic alterations, such as loss of methylation at KvDMR1, gain of methylation at H19-DMR, paternal uniparental disomy of chromosome 11, CDKN1C mutations, and structural abnormalities of chromosome 11. CDKN1C is an imprinted gene with maternal preferential expression, encoding for a cyclin-dependent kinase (CDK) inhibitor. Mutations in CDKN1C are found in 40 % of familial BWS cases with dominant maternal transmission and in ~5 % of sporadic cases. In this study, we searched for CDKN1C mutations in 37 BWS cases that had no evidence for other alterations. We found five mutations—four novel and one known—from a total of six patients. Four were maternally inherited and one was a de novo mutation. Two frame-shift mutations and one nonsense mutation abolished the QT domain, containing a PCNA-binding domain and a nuclear localization signal. Two missense mutations occurred in the CDK inhibitory domain, diminishing its inhibitory function. The above-mentioned mutations were predicted by in silico analysis to lead to loss of function; therefore, we strongly suspect that such anomalies are causative in the etiology of BWS.     

Characterization of differentially methylated regions in 3 bovine imprinted genes: a model for studying human germ-cell and embryo development

Correct imprinting is crucial for normal fetal and placental development in mammals. Experimental evidence in animal models and epidemiological studies in humans suggest that assisted reproductive technologies (ARTs) can interfere with imprinted gene regulation in gametogenesis and early embryogenesis. Bos taurus is an agriculturally important species in which ARTs are commonly employed. Because this species exhibits a similar preimplantation development and gestation length as humans, it is increasingly being used as a model for human germ-cell and embryo development. However, in contrast to humans and mice, there is relatively little information on bovine imprinted genes. Here, we characterized the bovine intergenic IGF2-H19 imprinting control region (ICR) spanning approximately 3 kb. We identified a 300-bp differentially methylated region (DMR) approximately 6 kb upstream of the H19 promoter, containing a CpG island with CTCF-binding site and high sequence similarity with the human intergenic ICR. Additional differentially methylated CpG islands lie -6 kb to -3 kb upstream of the promoter, however these are less conserved. Both classical bisulfite sequencing and bisulfite pyrosequencing demonstrated complete methylation of the IGF2-H19 ICR in sperm, complete demethylation in parthenogenetic embryos having only the female genome, and differential methylation in placental and somatic tissues. In addition, we established pyrosequencing assays for the previously reported bovine SNRPN and PEG3 DMRs. The observed methylation patterns were consistent with genomic imprinting in all analyzed tissues/cell types. The identified IGF2-H19 ICR and the developed quantitative methylation assays may prove useful for further studies on the relationship between ARTs and imprinting defects in the bovine model.     

A dyad oct-binding sequence functions as a maintenance sequence for the unmethylated state within the H19/Igf2-imprinted control region

DNA methylation of an imprinted control region (ICR) directs the allele-specific and reciprocal expression of the mouse H19 and the insulin-like growth factor 2 (Igf2) genes, mediated by controlling enhancer access. The ICR shows enhancer blocking activity through CTCF binding to an unmethylated sequence. The unmethylated state of the maternal ICR is maintained throughout development after establishment in the germ line; however, little is known of the molecular mechanisms that regulate DNA methylation. Hence, in this study we show that a dyad Oct-binding sequence (DOS) in the ICR mediates the demethylation of low-density methylation but not hypermethylation and is required to maintain the unmethylated state against the tendency for de novo methylation within the ICR in the embryonic carcinoma cell line P19. Furthermore, we also reveal that the unmethylated state of at least one CTCF-binding site within the ICR is under the control of DOS. Our results suggest that the ICR, as a CTCF-dependent insulator, requires DOS as well as CTCF-binding sites and that DOS maintains the maternal specific unmethylated state of the ICR at postimplantation stages.     

Alterations of H19 Imprinting and IGF2 Replication Timing Are Infrequent in Beckwith–Wiedemann Syndrome

Beckwith–Wiedemann syndrome (BWS) is an overgrowth disorder resulting from dysregulation of multiple imprinted genes through a variety of distinct mechanisms. A frequent alteration in BWS involves changes in the imprinting status of the coordinately regulated IGF2 and H19 genes on 11p15. Patients have been categorized according to alterations in the imprinted expression, allele-specific methylation, and regional replication timing of these genes. In this work, IGF2/H19 expression, H19 DNA methylation, and IGF2 regional replication timing were studied in nine karyotypically normal BWS fibroblasts and two BWS patients with maternally inherited 11p15 chromosomal rearrangements. Informative patients (9/9) maintained normal monoallelic H19 expression/methylation, despite biallelic IGF2 expression in 6/9. Replication timing studies revealed no changes in the pattern of asynchronous replication timing for both a patient with biallelic IGF2 expression and a patient carrying an 11p15 inversion. In contrast, a patient with a chromosome 11;22 translocation and normal H19 expression/methylation exhibited partial loss of asynchrony and a shift toward earlier replication times. These results indicate that in BWS, (1) H19 imprinting alterations are less frequent than previously estimated, (2) IGF2 imprinting and H19 imprinting are not necessarily coordinated, and (3) alterations in regional replication timing are generally not correlated with either chromosomal rearrangements or the imprinting status of IGF2 and H19.     

Comparison of bisulfite sequencing PCR with pyrosequencing for measuring differences in DNA methylation

DNA methylation strongly affects chromatin structure and the regulation of gene expression. For many years, bisulfite sequencing PCR (BSP) has served as the “gold standard” for measuring DNA methylation. However, with the evolution of pyrosequencing as a tool to evaluate DNA methylation, the need arises to compare the relative efficiencies of the two techniques in measuring DNA methylation. We provide for the first time a direct assessment of BSP and pyrosequencing to detect and quantify hypomethylation, hypermethylation, and mixed methylation of the ABCB1 promoter in various drug-sensitive and drug-resistant MCF-7 breast cancer cell lines through head-to-head experimentation. Our findings indicate that although both methods can reliably detect increased, decreased, and mixed methylation of DNA, BSP appears to be more sensitive than pyrosequencing at detecting strong hypermethylation of DNA. However, we also observed greater variability in the methylation of CpG sites by BSP, possibly due to the additional bacterial cloning step required by BSP over pyrosequencing. BSP and pyrosequencing equally detected hypomethylation and mixed methylation of DNA. The ability of pyrosequencing to reliably detect differences in DNA methylation across cell populations without requiring the cloning of bisulfite-treated DNA into bacterial expression vectors was seen as a major advantage of this technique.     

Children with idiopathic hemihypertrophy and beckwith-wiedemann syndrome have different constitutional epigenotypes associated with wilms tumor

Idiopathic hemihypertrophy (IH) is a congenital overgrowth syndrome associated with an increased risk of embryonal cancers in childhood. A related developmental disorder is Beckwith-Wiedemann syndrome (BWS), which increases risk for embryonal cancers, including Wilms tumor. Constitutional epigenetic alterations associated with BWS have been well characterized and include epigenetic alterations of imprinted genes on 11p15. The frequency of hypermethylation of H19 in children with IH and Wilms tumor, 20% (3/15), was significantly lower than the frequency in children with BWS and Wilms tumor, 79% (11/14; P = .0028). These results indicate that children with IH and Wilms tumor have different constitutional epigenotypes from those of children with BWS and Wilms tumor.     

Loss of CpG methylation is strongly correlated with loss of histone H3 lysine 9 methylation at DMR-LIT1 in patients with Beckwith-Wiedemann syndrome

To clarify the chromatin-based imprinting mechanism of the p57(KIP2)/LIT1 subdomain at chromosome 11p15.5 and the mouse ortholog at chromosome 7F5, we investigated the histone-modification status at a differentially CpG methylated region of Lit1/LIT1 (DMR-Lit1/LIT1), which is an imprinting control region for the subdomain and is demethylated in half of patients with Beckwith-Wiedemann syndrome (BWS). Chromatin-immunoprecipitation assays revealed that, in both species, DMR-Lit1/LIT1 with the CpG-methylated, maternally derived inactive allele showed histone H3 Lys9 methylation, whereas the CpG-unmethylated, paternally active allele was acetylated on histone H3/H4 and methylated on H3 Lys4. We have also investigated the relationship between CpG methylation and histone H3 Lys9 methylation at DMR-LIT1 in patients with BWS. In a normal individual and in patients with BWS with normal DMR-LIT1 methylation, histone H3 Lys9 methylation was detected on the maternal allele; however, it disappeared completely in the patients with the DMR-LIT1 imprinting defect. These findings suggest that the histone-modification status at DMR-Lit1/LIT1 plays an important role in imprinting control within the subdomain and that loss of histone H3 Lys9 methylation, together with CpG demethylation on the maternal allele, may lead to the BWS phenotype.     

Distinct methylation changes at the IGF2-H19 locus in congenital growth disorders and cancer

Background

Differentially methylated regions (DMRs) are associated with many imprinted genes. In mice methylation at a DMR upstream of the H19 gene known as the Imprint Control region (IC1) is acquired in the male germline and influences the methylation status of DMRs 100 kb away in the adjacent Insulin-like growth factor 2 (Igf2) gene through long-range interactions. In humans, germline-derived or post-zygotically acquired imprinting defects at IC1 are associated with aberrant activation or repression of IGF2, resulting in the congenital growth disorders Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, respectively. In Wilms tumour and colorectal cancer, biallelic expression of IGF2 has been observed in association with loss of methylation at a DMR in IGF2. This DMR, known as DMR0, has been shown to be methylated on the silent maternal IGF2 allele presumably with a role in repression. The effect of IGF2 DMR0 methylation changes in the aetiology of BWS or SRS is unknown.

Methodology/Principal Findings

We analysed the methylation status of the DMR0 in BWS, SRS and Wilms tumour patients by conventional bisulphite sequencing and pyrosequencing. We show here that, contrary to previous reports, the IGF2 DMR0 is actually methylated on the active paternal allele in peripheral blood and kidney. This is similar to the IC1 methylation status and is inconsistent with the proposed silencing function of the maternal IGF2 allele. Beckwith-Wiedemann and Silver-Russell patients with IC1 methylation defects have similar methylation defects at the IGF2 DMR0, consistent with IC1 regulating methylation at IGF2 in cis. In Wilms tumour, however, methylation profiles of IC1 and IGF2 DMR0 are indicative of methylation changes occurring on both parental alleles rather than in cis.

Conclusions/Significance

These results support a model in which DMR0 and IC1 have opposite susceptibilities to global hyper and hypomethylation during tumorigenesis independent of the parent of origin imprint. In contrast, during embryogenesis DMR0 is methylated or demethylated according to the germline methylation imprint at the IC1, indicating different mechanisms of imprinting loss in neoplastic and non-neoplastic cells.     

Assessment of p57(KIP2) gene mutation in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder involving developmental anomalies, tissue and organ hyperplasia and an increased risk of embryonic tumours (most commonly Wilms' tumour). This multigenic disorder is caused by dysregulation of the expression of imprinted genes in the 11p15 chromosomal region. It may involve paternal uniparental disomy (UPD), loss of imprinting of the IGF2 gene, maternal inherited translocations and trisomy with paternal duplication. Recently, a small proportion of BWS patients has been shown to have a mutation in the paternal imprinted p57(KIP2) gene, which encodes a cyclin-dependent kinase inhibitor and negatively regulates cell proliferation. We screened for p57(KIP2) gene mutations in 21 BWS patients with no 11p15 UPD in leucocyte DNA. All patients had a phenotype typical of BWS. We analysed the entire coding sequence of p57(KIP2), including intron-exon boundaries, by direct sequencing of five PCR-amplified fragments. No mutation was found in the p57(KIP2) gene. Our results are consistent with those of previous studies showing that mutation of p57(KIP2) is infrequent in BWS. Thus, other mechanisms of p57(KIP2) silencing (imprinting errors) and/or other 11p15 genes are probably involved in the pathogenesis of BWS.