Agostina De Crescenzo

Hypomethylation at multiple maternally methylated imprinted regions including PLAGL1 and GNAS loci in Beckwith–Wiedemann syndrome

Genomic imprinting is an epigenetic phenomenon restricting gene expression in a manner dependent on parent of origin. Imprinted gene products are critical regulators of growth and development, and imprinting disorders are associated with both genetic and epigenetic mutations, including disruption of DNA methylation within the imprinting control regions (ICRs) of these genes. It was recently reported that some patients with imprinting disorders have a more generalised imprinting defect, with hypomethylation at a range of maternally methylated ICRs. We report a cohort of 149 patients with a clinical diagnosis of Beckwith–Wiedemann syndrome (BWS), including 81 with maternal hypomethylation of the KCNQ1OT1 ICR. Methylation analysis of 11 ICRs in these patients showed that hypomethylation affecting multiple imprinted loci was restricted to 17 patients with hypomethylation of the KCNQ1OT1 ICR, and involved only maternally methylated loci. Both partial and complete hypomethylation was demonstrated in these cases, suggesting a possible postzygotic origin of a mosaic imprinting error. Some ICRs, including the PLAGL1 and GNAS/NESPAS ICRs implicated in the aetiology of transient neonatal diabetes and pseudohypoparathyroidism type 1b, respectively, were more frequently affected than others. Although we did not find any evidence for mutation of the candidate gene DNMT3L, these results support the hypotheses that trans-acting factors affect the somatic maintenance of imprinting at multiple maternally methylated loci and that the clinical presentation of these complex cases may reflect the loci and tissues affected with the epigenetic abnormalities.

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.

The KCNQ1OT1 Imprinting Control Region and non-coding RNA: new properties derived from the study of Beckwith-Wiedemann syndrome and Silver-Russell syndrome cases

A cluster of imprinted genes at chromosome 11p15.5 is associated with the growth disorders, Silver–Russell syndrome (SRS) and Beckwith–Wiedemann syndrome (BWS). The cluster is divided into two domains with independent imprinting control regions (ICRs). We describe two maternal 11p15.5 microduplications with contrasting phenotypes. The first is an inverted and in cis duplication of the entire 11p15.5 cluster associated with the maintenance of genomic imprinting and with the SRS phenotype. The second is a 160 kb duplication also inverted and in cis, but resulting in the imprinting alteration of the centromeric domain. It includes the centromeric ICR (ICR2) and the most 5′ 20 kb of the non-coding KCNQ1OT1 gene. Its maternal transmission is associated with ICR2 hypomethylation and the BWS phenotype. By excluding epigenetic mosaicism, cell clones analysis indicated that the two closely located ICR2 sequences resulting from the 160 kb duplication carried discordant DNA methylation on the maternal chromosome and supported the hypothesis that the ICR2 sequence is not sufficient for establishing imprinted methylation and some other property, possibly orientation-dependent, is needed. Furthermore, the 1.2 Mb duplication demonstrated that all features are present for correct imprinting at ICR2 when this is duplicated and inverted within the entire cluster. In the individuals maternally inheriting the 160 kb duplication, ICR2 hypomethylation led to the expression of a truncated KCNQ1OT1 transcript and to down-regulation of CDKN1C. We demonstrated by chromatin RNA immunopurification that the KCNQ1OT1 RNA interacts with chromatin through its most 5′ 20 kb sequence, providing a mechanism likely mediating the silencing activity of this long non-coding RNA.

The molecular function and clinical phenotype of partial deletions of the IGF2/H19 imprinting control region depends on the spatial arrangement of the remaining CTCF-binding sites

At chromosome 11p15.5, the imprinting centre 1 (IC1) controls the parent of origin-specific expression of the IGF2 and H19 genes. The 5 kb IC1 region contains multiple target sites (CTS) for the zinc-finger protein CTCF, whose binding on the maternal chromosome prevents the activation of IGF2 and allows that of H19 by common enhancers. CTCF binding helps maintaining the maternal IC1 methylation-free, whereas on the paternal chromosome gamete-inherited DNA methylation inhibits CTCF interaction and enhancer-blocking activity resulting in IGF2 activation and H19 silencing. Maternally inherited 1.4–2.2 kb deletions are associated with methylation of the residual CTSs and Beckwith–Wiedemann syndrome, although with different penetrance and expressivity. We explored the relationship between IC1 microdeletions and phenotype by analysing a number of previously described and novel mutant alleles. We used a highly quantitative assay based on next generation sequencing to measure DNA methylation in affected families and analysed enhancer-blocking activity and CTCF binding in cultured cells. We demonstrate that the microdeletions mostly affect IC1 function and CTCF binding by changing CTS spacing. Thus, the extent of IC1 inactivation and the clinical phenotype are influenced by the arrangement of the residual CTSs. A CTS spacing similar to the wild-type allele results in moderate IC1 inactivation and is associated with stochastic DNA methylation of the maternal IC1 and incomplete penetrance. Microdeletions with different CTS spacing display severe IC1 inactivation and are associated with IC1 hypermethylation and complete penetrance. Careful characterization of the IC1 microdeletions is therefore needed to predict recurrence risks and phenotypical outcomes.

(Epi)genotype–phenotype correlations in Beckwith–Wiedemann syndrome

Beckwith–Wiedemann syndrome (BWS) is characterized by cancer predisposition, overgrowth and highly variable association of macroglossia, abdominal wall defects, nephrourological anomalies, nevus flammeus, ear malformations, hypoglycemia, hemihyperplasia, and organomegaly. BWS molecular defects, causing alteration of expression or activity of the genes regulated by two imprinting centres (IC) in the 11p15 chromosomal region, are also heterogeneous. In this paper we define (epi)genotype–phenotype correlations in molecularly confirmed BWS patients. The characteristics of 318 BWS patients with proven molecular defect were compared among the main four molecular subclasses: IC2 loss of methylation (IC2-LoM, n=190), IC1 gain of methylation (IC1-GoM, n=31), chromosome 11p15 paternal uniparental disomy (UPD, n=87), and cyclin-dependent kinase inhibitor 1C gene (CDKN1C) variants (n=10). A characteristic growth pattern was found in each group; neonatal macrosomia was almost constant in IC1-GoM, postnatal overgrowth in IC2-LoM, and hemihyperplasia more common in UPD (P<0.001). Exomphalos was more common in IC2/CDKN1C patients (P<0.001). Renal defects were typical of UPD/IC1 patients, uretheral malformations of IC1-GoM cases (P<0.001). Ear anomalies and nevus flammeus were associated with IC2/CDKN1C genotype (P<0.001). Macroglossia was less common among UPD patients (P<0.001). Wilms’ tumor was associated with IC1-GoM or UPD and never observed in IC2-LoM patients (P<0.001). Hepatoblastoma occurred only in UPD cases. Cancer risk was lower in IC2/CDKN1C, intermediate in UPD, and very high in IC1 cases (P=0.009). In conclusion, (epi)genotype–phenotype correlations define four different phenotypic BWS profiles with some degree of clinical overlap. These observations impact clinical care allowing to move toward (epi) genotype-based follow-up and cancer screening.

Nephrological findings and genotype–phenotype correlation in Beckwith–Wiedemann syndrome

Beckwith–Wiedemann syndrome (BWS), an overgrowth disorder with several congenital abnormalities, encompasses nephrourological anomalies. The objective of the report is to analyze the latter and related genotype–phenotype correlations. The study was a retrospective review of nephrourological investigations and genotype in 67 BWS patients. Imaging and laboratory studies have been correlated with the molecular anomalies typical of BWS. Thirty-eight (56.7%) patients had a total of 61 nonmalignant nephrourological findings, including nephromegaly (n = 24), collecting system abnormalities (n = 14), cryptorchidism (n = 11), nephrolithiasis (n = 5), cysts (n = 5), and dysplasia (n = 1). Four patients had Wilms’ tumor, all associated with renal hyperplasia. Renal findings were almost consistent in the BWSIC1 group, with nephromegaly in all patients and collecting system abnormalities in half of them. BWSUPD and negative patients also had frequent anomalies (63.6% and 61.9% respectively), whereas only 36.0% of BWSIC2 had renal findings (p = 0.003). Cryptorchidism was associated with abdominal wall defects (p < 0.001) appearing more frequently in BWSIC2 (p = 0.028). Urinary tract infections were observed in 17.9% of patients, with two resulting in life-threatening sepsis. Hypercalciuria was present in 10% of cases. 55.5% of BWS patients have renal findings. Although variegate, these anomalies disclose a genotype–phenotype correlation.

Silver-Russell syndrome and Beckwith-Wiedemann syndrome phenotypes associated with 11p duplication in a single family.

Genomic imprinting is an epigenetic phenomenon resulting in differential expression of maternal and paternal alleles of a subset of genes. In the mouse, mutation of imprinted genes often results in contrasting phenotypes, depending on parental origin. The overgrowth-associated Beckwith-Wiedemann syndrome (BWS) and the growth restriction-associated Silver-Russell syndrome (SRS) have been linked with a variety of epigenetic and genetic defects affecting a cluster of imprinted genes at chromosome 11p15.5. Paternally derived and maternally derived 11p15.5 duplications represent infrequent findings in BWS and SRS, respectively. Here, we report a case in which a 6.5 Mb duplication of 11p15.4-pter resulted in SRS and BWS phenotypes in a child and her mother, respectively. Molecular analyses demonstrated that the duplication involved the maternal chromosome 11p15 in the child and the paternal chromosome 11p15 in the mother. This observation provides a direct demonstration that SRS and BWS represent specular images, both at the clinical and molecular levels.

A case of Beckwith-Wiedemann syndrome caused by a cryptic 11p15 deletion encompassing the centromeric imprinted domain of the BWS locus.

Background Beckwith–Wiedemann syndrome (BWS) is a clinically variable and genetically heterogeneous disorder, providing evidence that imprinted genes play key roles in the control of fetal growth. Clinically, diagnostic criteria include macrosomia, macroglossia, abdominal wall defects, neonatal hypoglycaemia, visceromegalies and hemihyperplasia. Component clinical manifestations also include renal abnormalities, adrenocortical cytomegaly and a characteristic facial appearance, with midface hypoplasia and ear anomalies. Genetically, BWS is associated with disturbances within two different domains on 11p15 that are controlled by distinct imprinting control regions (ICR), ICR1 and ICR2. The majority of patients have abnormalities within ICR2. In particular, loss of maternal methylation accounts for 50–60% of cases, and is associated with reduction in the expression of the CDKN1C gene, a member of the cyclin dependent kinase inhibitor family acting as negative regulator of cell proliferation. Mutations in CDKN1C are detected in another 5–10% of subjects with sporadic BWS. Chromosome deletions affecting ICR2 are uncommon. Methods and findings We report on a patient with BWS in which a de novo 11p15 deletion was detected by array comparative genomic hybridisation. Clinically, the patient presented with mild mental retardation and minor physical anomalies. The deletion, that was demonstrated to be maternal in origin by SNP array, encompassed ICR2 and several flanking genes, including CDKN1C. A normal methylation pattern of ICR1 was observed. Conclusions This observation provides evidence that, among the genetic defects associated with BWS, a 11p15 microdeletion encompassing ICR2 identifies a peculiar clinical phenotype, with high recurrence risk in offspring of female carriers. It also supports the model of two independent domains within the BWS locus.

Prevalence of beckwith–wiedemann syndrome in North West of Italy

Although Beckwith–Wiedemann syndrome (BWS, OMIM #130650) is the most common genetic overgrowth disorder, data on its epidemiology are scanty and the estimates of its occurrence show wide variability. The aim of this study is to assess its prevalence in Piedmont Region (Italy). We included in the study all patients diagnosed with BWS born in Piedmont from 1997 to 2009 through a search in the Italian Registry for Rare Diseases. This source was further validated with data from the network of Regional Clinical Genetics services and surveys in extra‐regional Clinical Genetics centres, laboratories and the Italian BWS patients association. All cases were further ascertained through physical exam, medical history and specific molecular tests. The search identified 46 clear‐cut cases of BWS born across the 13‐year period, providing a prevalence of 1:10 340 live births (95% confidence interval 1:7,752–13,698 live births). Among the 41 patients who underwent molecular tests, 70.7% were positive, showing hypomethylation of the IC2 imprinting center (29.3%), paternal chromosome 11 uniparental disomy (pUPD11, 24.4%), IC1 hypermethylation (14.6%), CDKN1c mutation (2.4%), whereas 29.3% had negative molecular tests. The study provides an approximate BWS prevalence of 1:10,000 live birth, the highest reported to date.

Silver-russell syndrome following in vitro fertilization

To the Editor, A number of observations have revealed an increased frequency of genomic imprinting disorders among children born using assisted reproduction technology (ART), pointing to a relationship between these procedures and methylation defects. It has been speculated that these defects result from interference of the ART procedures with the establishment or maintenance of imprinting through different stages of development. Based on analysis of the Beckwith-Wiedemann syndrome (MIM 130650) cases born after in vitro fertilization (IVF), a relative risk of 4.2 has been estimated, although this figure could well be biased by the patients’ recruitment criteria [1]. Silver-Russell Syndrome (SRS [MIM 180860]) is a clinically and genetically heterogeneous condition of primordial growth retardation that spares head growth; in approximately half of the cases, it causes a growth asymmetry in the limbs, trunk, or face [2]. We report a patient with SRS born after IVF, characterized by molecular testing. He was the only son of nonconsaguineous Caucasian parents. The 38-year-old mother and the 42year-old father had opted for IVF because of unilateral fallopian tube closure. Maternal oocytes taken after gonadotropin stimulation were mixed with paternal sperm collected using a condom. Ten fertilized ova were obtained, and 2 of them were transferred to the maternal uterus. A single living fetus was evidenced by standard ultrasound scan (US) at 7 weeks of gestation (WG). No exposure to teratogenic agents during pregnancy was reported. Serologic tests for cytomegalovirus, toxoplasmosis, and rubella and triple serum marker screening were negative. Amniocentesis at 16 WG revealed a normal male karyotype. A standard US at 22 WG disclosed a development corresponding to 18 weeks without evidence of fetal distress. The newborn was delivered by cesarean section because of breech presentation at 37 WG. Birth weight was 2050 g (,3rd centile), length 42.5 cm (3rd centile), and occipitalfrontal circumference (OFC) 34.4 cm (75th centile). Physical examination at 4 months disclosed an OFC of 42 cm (50th centile), length 51.5 cm, and weight 4330 g (both ,3rd centile) with mild asymmetry and left hemimacrosoma resulting in a discrepancy of 1.5 cm in leg length (Fig. 1a). There were mild facial dysmorphisms (Fig. 1b,c) and bilateral 5th finger clinodactyly (Fig. 1d). The infant had feeding difficulties, which were treated with multiple meals and required nasogastric and gastrostomy tube feeding. At 13 months the weight was 10.200 kg (50th centile), length 74.7 cm (10th to 25th centile), and OFC 49 cm (90th centile). He walked unaided at the age of 15 months. Thyroid, growth hormone, and insuline-like growth hormone-1 hormonal tests were normal. Analysis of DNA methylation of the chromosome 11p15.5 imprinted gene cluster by bisulfite treatment coupled with restriction enzyme digestion (methylation restriction-polymerase chain reaction or combined bisulfate restriction analysis), disclosed a partial hypomethylation of the Imprinting Center 1 (IC1) (Fig. 2) [3]. (Supplemental material on the methods utilized is available online.) This is an additional example of a patient with a methylation defect conceived with ART. Like most SRS patients that present asymmetric growth, he carries an epimutation of the 11p15.5 region. The partial rather than complete loss of methylation of the paternal IC1 in these patients suggests that the asymmetric growth could result from a mosaic distribution of a postfertilization error. Given the variable SRS phenotype and a reported 2.6 relative risk of low birth weight unrelated to prematurity and twin gestations in the IVF population, a number of these smallfor-gestational-age infants within this population could represent undiagnosed cases of SRS.