Analysis of germline CDKN1C (p57KIP2) mutations in familial and sporadic Beckwith-Wiedemann syndrome (BWS) provides a novel genotype-phenotype correlation.

Beckwith-Wiedemann syndrome (BWS) is a human imprinting disorder with a variable phenotype. The major features are anterior abdominal wall defects including exomphalos (omphalocele), pre- and postnatal overgrowth, and macroglossia. Additional less frequent complications include specific developmental defects and a predisposition to embryonal tumours. BWS is genetically heterogeneous and epigenetic changes in the IGF2/H19 genes resulting in overexpression of IGF2 have been implicated in many cases. Recently germline mutations in the cyclin dependent kinase inhibitor gene CDKN1C (p57KIP2) have been reported in a variable minority of BWS patients. We have investigated a large series of familial and sporadic BWS patients for evidence of CDKN1C mutations by direct gene sequencing. A total of 70 patients with classical BWS were investigated; 54 were sporadic with no evidence of UPD and 16 were familial from seven kindreds. Novel germline CDKN1C mutations were identified in five probands, 3/7 (43%) familial cases and 2/54 (4%) sporadic cases. There was no association between germline CDKN1C mutations and IGF2 or H19 epigenotype abnormalities. The clinical phenotype of 13 BWS patients with germline CDKN1C mutations was compared to that of BWS patients with other defined types of molecular pathology. This showed a significantly higher frequency of exomphalos in the CDKN1C mutation cases (11/13) than in patients with an imprinting centre defect (associated with biallelic IGF2 expression and H19 silencing) (0/5, p<0.005) or patients with uniparental disomy (0/9, p<0.005). However, there was no association between germline CDKN1C mutations and risk of embryonal tumours. No CDKN1C mutations were identified in six non-BWS patients with overgrowth and Wilms tumour. These findings (1) show that germline CDKN1C mutations are a frequent cause of familial but not sporadic BWS, (2) suggest that CDKN1C mutations probably cause BWS independently of changes in IGF2/H19 imprinting, (3) provide evidence that aspects of the BWS phenotype may be correlated with the involvement of specific imprinted genes, and (4) link genotype-phenotype relationships in BWS and the results of murine experimental models of BWS.     

Epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is a model imprinting disorder resulting from mutations or epigenetic events involving imprinted genes at chromosome 11p15.5. Thus, germline mutations in CDKN1C, uniparental disomy (UPD), and loss of imprinting of IGF2 and other imprinted genes have been implicated. Many familial BWS cases have germline CDKN1C mutations. However, most BWS cases are sporadic and UPD or putative imprinting errors predominate in this group. We have identified previously a subgroup of sporadic cases with loss of imprinting (LOI) of IGF2 and epigenetic silencing of H19 proposed to be caused by a defect in a distal 11p15.5 imprinting control element (designated BWSIC1). However, many sporadic BWS patients show biallelic IGF2 expression in the presence of normal H19 methylation and expression patterns. This and other evidence suggested the existence of a further imprinting control element (BWSIC2) at 11p15.5. Recently, we showed that a subgroup of BWS patients have loss of methylation (LOM) at a differentially methylated region (KvDMR1) within the KCNQ1 gene centromeric to the IGF2 and H19 genes. We have now analysed a large series of sporadic cases to define the frequency and phenotypic correlates of epigenetic abnormalities in BWS. LOM at KvDMR1 was detected by Southern analysis or a novel PCR based method in 35 of 69 (51%) sporadic BWS without UPD. LOM at KvDMR1 was often, but not invariably associated with LOI of IGF2. KvDMR1 LOM was not detected in BWS patients with putative BWSIC1 defects and cases with KvDMR1 LOM (that is, putative BWSIC2 defects) invariably had a normal H19 methylation pattern. The incidence of exomphalos in putative BWSIC2 defect patients was not significantly different from that in patients with germline CDKN1C mutations (20/29 and 13/15 respectively), but was significantly greater than that in patients with putative BWSIC1 defects (0/5, p=0.007) and UPD (0/22, p<0.0001). These findings are consistent with the hypothesis that LOM of KvDMR1 (BWSIC2 defect) results in epigenetic silencing of CDKN1C and variable LOI of IGF2. BWS patients with embryonal tumours have UPD or a BWSIC1 defect but not LOM of KvDMR1. This study has further shown how (1) variations in phenotypic expression of BWS may be linked to specific molecular subgroups and (2) molecular analysis of BWS can provide insights into mechanisms of imprinting regulation.


Keywords: Beckwith-Wiedemann syndrome; epigenotype-phenotype correlations; imprinting     

Alternative mechanisms associated with silencing of CDKN1C in Beckwith-Wiedemann syndrome

Background: Mutations in the imprinted gene CDKN1C account for approximately 10% of Beckwith–Wiedemann syndrome (BWS) cases. Fibroblasts from BWS patients with loss of methylation (LOM) at the imprinting control region (ICR) KvDMR1 have reduced CDKN1C expression. Another group of BWS patients with downregulated CDKN1C expression but with normal methylation at KvDMR1 has been identified. Objective: To investigate the mechanism of CDKN1C silencing in BWS in these two classes of patients. Methods: The CDKN1C promoter region was analysed for changes in DNA methylation using bisulphite sequencing, and for alterations in chromatin structure using the chromatin immunoprecipitation (ChIP) assay. Results: There was only spurious CpG methylation of the CDKN1C promoter in fibroblast DNA from both normal individuals and patients with BWS, irrespective of the methylation status of KvDMR1. There was no detectable change in chromatin structure at the CDKN1C promoter in patients with LOM at KvDMR1. BWS patients with downregulated CDKN1C and normal methylation at KvDMR1 had depletion of dimethylated H3-K4 and enrichment of dimethylated H3-K9 and HP1γ at the CDKN1C promoter, suggesting that in these cases gene silencing is associated with repressive chromatin changes. Conclusions: CDKN1C may be downregulated by multiple mechanisms including some that do not involve promoter methylation. In BWS patients with normal methylation at KvDMR1 and reduced expression of CDKN1C, repressive chromatin may play a role, but the absence of methylation and repressive chromatin structure at the CDKN1C promoter in BWS patients with LOM at KvDMR1 argues for a direct role of this epimutation in silencing CDKN1C.     

Familial Beckwith-Wiedemann syndrome in a multigenerational family: Forty years of careful phenotyping

Beckwith-Wiedemann Spectrum (BWSp) is an overgrowth and cancer predisposition disorder characterized by a wide spectrum of phenotypic manifestations including macroglossia, abdominal wall defects, neonatal hypoglycemia, and predisposition to embryonal tumors. In 1981, Best and Hoekstra reported four patients with BWSp in a single family which suggested autosomal dominant inheritance, but standard clinical testing for BWSp was not available during this time. Meticulous phenotyping of this family has occurred over the past 40 years of follow-up with additional family members being identified and samples collected for genetic testing. Genetic testing revealed a pathogenic mutation in CDKN1C, consistent with the most common cause of familial BWSp. CDKN1C mutations account for just 5% of sporadic cases of BWSp. Here, we report the variable presentation of BWSp across the individuals affected by the CDKN1C mutation and other extended family members spanning multiple generations, all examined by the same physician. Additional phenotypes thought to be atypical in patients with BWSp were reported which included cardiac abnormalities. The incidence of tumors was documented in extended family members and included rhabdomyosarcoma, astrocytoma, and thyroid carcinoma, which have previously been reported in patients with BWSp. These observations suggest that in addition to the inheritance of the CDKN1C variant, there are modifying factors in this family driving the phenotypic spectrum observed. Alternative theories are suggested to explain the etiology of clinical variability including diffused mosaicism, anticipation, and the presence of additional variants tracking in the family. This study highlights the necessity of long-term follow-up in patients with BWSp and consideration of individual familial characteristics in the context of phenotype and/or (epi)genotype associations.     

Silencing of CDKN1C (p57KIP2) is associated with hypomethylation at KvDMR1 in Beckwith-Wiedemann syndrome

Context: Beckwith–Wiedemann syndrome (BWS) arises by several genetic and epigenetic mechanisms affecting the balance of imprinted gene expression in chromosome 11p15.5. The most frequent alteration associated with BWS is the absence of methylation at the maternal allele of KvDMR1, an intronic CpG island within the KCNQ1 gene. Targeted deletion of KvDMR1 suggests that this locus is an imprinting control region (ICR) that regulates multiple genes in 11p15.5. Cell culture based enhancer blocking assays indicate that KvDMR1 may function as a methylation modulated chromatin insulator and/or silencer.

Objective: To determine the potential consequence of loss of methylation (LOM) at KvDMR1 in the development of BWS.

Methods: The steady state levels of CDKN1C gene expression in fibroblast cells from normal individuals, and from persons with BWS who have LOM at KvDMR1, was determined by both real time quantitative polymerase chain reaction (qPCR) and ribonuclease protection assay (RPA). Methylation of the CDKN1C promoter region was assessed by Southern hybridisation using a methylation sensitive restriction endonuclease.

Results: Both qPCR and RPA clearly demonstrated a marked decrease (86–93%) in the expression level of the CDKN1C gene in cells derived from patients with BWS, who had LOM at KvDMR1. Southern analysis indicated that downregulation of CDKN1C in these patients was not associated with hypermethylation at the presumptive CDKN1C promoter.

Conclusions: An epimutation at KvDMR1, the absence of maternal methylation, causes the aberrant silencing of CDKN1C, some 180 kb away on the maternal chromosome. Similar to mutations at this locus, this silencing may give rise to BWS.

     

Fetal growth patterns in Beckwith–Wiedemann syndrome

We provide data on fetal growth pattern on the molecular subtypes of Beckwith–Wiedemann syndrome (BWS): IC1 gain of methylation (IC1‐GoM), IC2 loss of methylation (IC2‐LoM), 11p15.5 paternal uniparental disomy (UPD), and CDKN1C mutation. In this observational study, gestational ages and neonatal growth parameters of 247 BWS patients were compared by calculating gestational age‐corrected standard deviation scores (SDS) and proportionality indexes to search for differences among IC1‐GoM (n = 21), UPD (n = 87), IC2‐LoM (n = 147), and CDKN1C mutation (n = 11) patients. In IC1‐GoM subgroup, weight and length are higher than in other subgroups. Body proportionality indexes display the following pattern: highest in IC1‐GoM patients, lowest in IC2‐LoM/CDKN1C patients, intermediate in UPD ones. Prematurity was significantly more prevalent in the CDKN1C (64%) and IC2‐LoM subgroups (37%). Fetal growth patterns are different in the four molecular subtypes of BWS and remarkably consistent with altered gene expression primed by the respective molecular mechanisms. IC1‐GoM cases show extreme macrosomia and severe disproportion between weight and length excess. In IC2‐LoM/CDKN1C patients, macrosomia is less common and associated with more proportionate weight/length ratios with excess of preterm birth. UPD patients show growth patterns closer to those of IC2‐LoM, but manifest a body mass disproportion rather similar to that seen in IC1‐GoM cases.     

A novel de novo point mutation of the OCT‐binding site in the IGF2/H19‐imprinting control region in a Beckwith–Wiedemann syndrome patient

The IGF2/H19‐imprinting control region (ICR1) functions as an insulator to methylation‐sensitive binding of CTCF protein, and regulates imprinted expression of IGF2 and H19 in a parental origin‐specific manner. ICR1 methylation defects cause abnormal expression of imprinted genes, leading to Beckwith–Wiedemann syndrome (BWS) or Silver–Russell syndrome (SRS). Not only ICR1 microdeletions involving the CTCF‐binding site, but also point mutations and a small deletion of the OCT‐binding site have been shown to trigger methylation defects in BWS. Here, mutational analysis of ICR1 in 11 BWS and 12 SRS patients with ICR1 methylation defects revealed a novel de novo point mutation of the OCT‐binding site on the maternal allele in one BWS patient. In BWS, all reported mutations and the small deletion of the OCT‐binding site, including our case, have occurred within repeat A2. These findings indicate that the OCT‐binding site is important for maintaining an unmethylated status of maternal ICR1 in early embryogenesis.     

Beckwith-Wiedemann syndrome and uniparental disomy 11p: fine mapping of the recombination breakpoints and evaluation of several techniques

Beckwith-Wiedemann syndrome (BWS) is a phenotypically and genotypically heterogeneous overgrowth syndrome characterized by somatic overgrowth, macroglossia and abdominal wall defects. Other usual findings are hemihyperplasia, embryonal tumours, adrenocortical cytomegaly, ear anomalies, visceromegaly, renal abnormalities, neonatal hypoglycaemia, cleft palate, polydactyly and a positive family history. BWS is a complex, multigenic disorder associated, in up to 90% of patients, with alteration in the expression or function of one or more genes in the 11p15.5 imprinted gene cluster. There are several molecular anomalies associated with BWS and the large proportion of cases, about 85%, is sporadic and karyotypically normal. One of the major categories of BWS molecular alteration (10-20% of cases) is represented by mosaic paternal uniparental disomy (pUPD), namely patients with two paternally derived copies of chromosome 11p15 and no maternal contribution for that. In these patients, in addition to the effects of IGF2 overexpression, a decreased level of the maternally expressed gene CDKN1C may contribute to the BWS phenotype. In this paper, we reviewed a series of nine patients with BWS because of pUPD using several methods with the aim to evaluate the percentage of mosaicism, the methylation status at both loci, the extension of the pUPD at the short arm and the breakpoints of recombination. Fine mapping of mitotic recombination breakpoints by single-nucleotide polymorphism-array in individuals with UPD and fine estimation of epigenetic defects will provide a basis for understanding the aetiology of BWS, allowing more accurate prognostic predictions and facilitating management and surveillance of individuals with this disorder.     

Analysis of CDKN1C in Beckwith Wiedemann syndrome

In this study we have examined 32 patients with Beckwith Wiedemann Syndrome (BWS) for mutations affecting the CDKN1C gene, including seven cases of familial BWS. Mutations were not detected in the coding region of the CDKN1C gene in any individual with BWS. However in two patients, two G/A base substitutions at adjacent positions in the 5'UTR were detected. These substitutions were also found in normal controls. Expression of CDKN1C in somatic tissues was examined in 18 of the 32 cases using semi-quantitative RT-PCR. CDKN1C expression was significantly reduced in the peripheral blood of three cases compared with controls. These results suggest that, although coding region mutations in the CDKN1C gene are rare in BWS, mutations disrupting CDKN1C expression may be found. Three of five informative patients exhibited biallelic CDKN1C expression in lymphocytes, cord blood, and kidney tissue, respectively. Biallelic expression was not associated with overall CDKN1C levels significantly different to those in controls. Patients who expressed CDKN1C biallelically, or who were low CDKN1C expressors, maintained monoallelic methylation in the Differentially Methylated Region 2 (DMR2) of the IGF2 locus. One patient expressing CDKN1C biallelically, maintained imprinted gene expression at the IGF2 locus. These results suggest that biallelic CDKN1C expression does not significantly perturb the overall levels of CDKN1C expression in somatic tissue. They also confirm other studies showing that the mechanisms associated with regulating CDKN1C expression and imprinting are separate from those regulating IGF2 imprinting.     

Brain abnormalities in patients with Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder with variability in clinical manifestations and molecular causes. In most cases, patients with BWS have normal development. Cases with developmental delay are usually attributed to neonatal hypoglycemia or chromosome abnormalities involving copy number variation for genes beyond the critical BWS region at 11p15.5. Brain abnormalities have not previously been recognized within the BWS phenotypic spectrum. We report on seven cases of BWS associated with posterior fossa abnormalities. Of these, two cases presented with Blake's pouch cyst, two with Dandy-Walker variant (DWV; hypoplasia of the inferior part of the vermis), one with Dandy-Walker malformation (DWM) and one with a complex of DWM, dysgenesis of the corpus callosum and brain stem abnormality. In all these cases, molecular findings involved the centromeric imprinted domain on chromosome locus 11p15.5, which includes imprinting center 2 (IC2) and the imprinted growth suppressor gene, CDKN1C. Three cases had loss of methylation at IC2, two had CDKN1C mutations, and one had loss of methylation at IC2 and a microdeletion. In one case no mutation/methylation abnormality was detected. These findings together with previously reported correlations suggest that genes in imprinted domain 2 at 11p15.5 are involved in normal midline development of several organs including the brain. Our data suggest that brain malformations may present as a finding within the BWS phenotype when the molecular etiology involves imprinted domain 2. Brain imaging may be useful in identifying such malformations in individuals with BWS and neurodevelopmental issues.     

New insights into the pathogenesis of Beckwith-Wiedemann and Silver-Russell syndromes: contribution of small copy number variations to 11p15 imprinting defects

The imprinted 11p15 region is organized in two domains, each of them under the control of its own imprinting control region (ICR1 for the IGF2/H19 domain and ICR2 for the KCNQ1OT1/CDKN1C domain). Disruption of 11p15 imprinting results in two fetal growth disorders with opposite phenotypes: the Beckwith-Wiedemann (BWS) and the Silver-Russell (SRS) syndromes. Various 11p15 genetic and epigenetic defects have been demonstrated in BWS and SRS. Among them, isolated DNA methylation defects account for approximately 60% of patients. To investigate whether cryptic copy number variations (CNVs) involving only part of one of the two imprinted domains account for 11p15 isolated DNA methylation defects, we designed a single nucleotide polymorphism array covering the whole 11p15 imprinted region and genotyped 185 SRS or BWS cases with loss or gain of DNA methylation at either ICR1 or ICR2. We describe herein novel small gain and loss CNVs in six BWS or SRS patients, including maternally inherited cis-duplications involving only part of one of the two imprinted domains. We also show that ICR2 deletions do not account for BWS with ICR2 loss of methylation and that uniparental isodisomy involving only one of the two imprinted domains is not a mechanism for SRS or BWS.     

p57Kip2 knock‐in mouse reveals CDK‐independent contribution in the development of Beckwith–Wiedemann syndrome

CDKN1C encodes the cyclin–CDK inhibitor p57^Kip2 (p57), a negative regulator of the cell cycle and putative tumour suppressor. Genetic and epigenetic alterations causing loss of p57 function are the most frequent cause of Beckwith–Wiedemann syndrome (BWS), a genetic disorder characterized by multiple developmental anomalies and increased susceptibility to tumour development during childhood. So far, BWS development has been attributed entirely to the deregulation of proliferation caused by loss of p57‐mediated CDK inhibition. However, a fraction of BWS patients have point mutations in CDKN1C located outside of the CDK inhibitory region, suggesting the involvement of other parts of the protein in the disease. To test this possibility, we generated knock‐in mice deficient for p57‐mediated cyclin–CDK inhibition (p57^CK–), the only clearly defined function of p57. Comparative analysis of p57^CK– and p57^KO mice provided clear evidence for CDK‐independent roles of p57 and revealed that BWS is not caused entirely by CDK deregulation, as several features of BWS are caused by the loss of CDK‐independent roles of p57. Thus, while the genetic origin of BWS is well understood, our results underscore that the underlying molecular mechanisms remain largely unclear. To probe these mechanisms further, we determined the p57 interactome. Several partners identified are involved in genetic disorders with features resembling those caused by CDKN1C mutation, suggesting that they could be involved in BWS pathogenesis and revealing a possible connection between seemingly distinct syndromes. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Molecular subtypes and phenotypic expression of Beckwith-Wiedemann syndrome

Beckwith-Wiedemann Syndrome (BWS) results from mutations or epigenetic events involving imprinted genes at 11p15.5. Most BWS cases are sporadic and uniparental disomy (UPD) or putative imprinting errors predominate in this group. Sporadic cases with putative imprinting defects may be subdivided into (a) those with loss of imprinting (LOI) of IGF2 and H19 hypermethylation and silencing due to a defect in a distal 11p15.5 imprinting control element (IC1) and (b) those with loss of methylation at KvDMR1, LOI of KCNQ1OT1 (LIT1) and variable LOI of IGF2 in whom there is a defect at a more proximal imprinting control element (IC2). We investigated genotype/epigenotype-phenotype correlations in 200 cases with a confirmed molecular genetic diagnosis of BWS (16 with CDKN1C mutations, 116 with imprinting centre 2 defects, 14 with imprinting centre 1 defects and 54 with UPD). Hemihypertrophy was strongly associated with UPD (P<0.0001) and exomphalos was associated with an IC2 defect or CDKN1C mutation but not UPD or IC1 defect (P<0.0001). When comparing birth weight centile, IC1 defect cases were significantly heavier than the patients with CDKN1C mutations or IC2 defect (P=0.018). The risk of neoplasia was significantly higher in UPD and IC1 defect cases than in IC2 defect and CDKN1C mutation cases. Kaplan-Meier analysis revealed a risk of neoplasia for all patients of 9% at age 5 years, but 24% in the UPD subgroup. The risk of Wilms' tumour in the IC2 defect subgroup appears to be minimal and intensive screening for Wilms' tumour appears not to be indicated. In UPD patients, UPD extending to WT1 was associated with renal neoplasia (P=0.054). These findings demonstrate that BWS represents a spectrum of disorders. Identification of the molecular subtype allows more accurate prognostic predictions and enhances the management and surveillance of BWS children such that screening for Wilms' tumour and hepatoblastoma can be focused on those at highest risk.     

Acute lymphocytic leukaemia in a child with Beckwith–Wiedemann syndrome harbouring a CDKN1C mutation

Beckwith–Wiedemann syndrome (BWS) is a rare overgrowth syndrome associated with an increased risk in childhood tumours. The phenotypic variability in BWS reflects its molecular heterogeneity. This syndrome is a multigenic disorder caused by dysregulation of imprinted growth regulatory genes in the 11p15.5 region. The most commonly reported tumours in this syndrome are tumours of embryologic origin such as Wilms tumours, hepatoblastomas, neuroblastomas, rhabdomyosarcomas and adrenocortical carcinomas.We report the case of a 10-year-old patient diagnosed with BWS, harbouring a CDKN1C (p57^KIP2) mutation, who developed a T-type acute lymphoblastic leukaemia.To our knowledge it is the first report of an acute lymphoblastic leukaemia of T-type in a child with BWS. We discuss the possibility of a link between BWS and leukaemia via one of the few known negative regulator of hematopoiesis, the transforming growth factor beta pathway, depending upon the up-regulation of CDKN1C.     

Investigation of a pervasive immune,cardiac, and behavioral phenotype in Beckwith-Wiedemann syndrome: A case report

Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder caused by genetic and epigenetic changes in the chromosome 11p15 region. The syndrome is characterized by a wide range of features including macrosomia, lateralized overgrowth, abdominal wall defects, and hypoglycemia. BWS presentation is variable across the entire patient population, but certain areas including immunology, cardiology, and behavioral differences are not well characterized. We present a case of a male patient with BWS due to the most common cause of BWS, loss of methylation at imprinting center 2 with a variable phenotype, including classical features (macrosomia, macroglossia, omphalocele, placentomegaly and mild lateralized overgrowth) in addition to uncommon features (immune deficiency, developmental delays, and pulmonary stenosis) not typically seen in BWS. This study defines a patient's clinical presentation and course and highlights the need to consider atypical organ systems in BWS as either an expansion of the phenotype or co-existing conditions to develop personalized care models.     

The role of germline AIP,MEN1, PRKAR1A,CDKN1B and CDKN2C mutations in causing pituitary adenomas in a large cohort of children,adolescents, and patients with genetic syndromes

Stratakis CA, Tichomirowa MA, Boikos S, Azevedo MF, Lodish M, Martari M, Verma S, Daly AF, Raygada M, Keil MF, Papademetriou J, Drori‐Herishanu L, Horvath A, Tsang KM, Nesterova M, Franklin S, Vanbellinghen J‐F, Bours V, Salvatori R, Beckers A. The role of germline AIP, MEN1, PRKAR1A, CDKN1B and CDKN2C mutations in causing pituitary adenomas in a large cohort of children, adolescents, and patients with genetic syndromes. The prevalence of germline mutations in MEN1, AIP, PRKAR1A, CDKN1B and CDKN2CI is unknown among pediatric patients with pituitary adenomas (PA). In this study, we screened children with PA for mutations in these genes; somatic GNAS mutations were also studied in a limited number of growth hormone (GH) or prolactin (PRL)‐secreting PA. We studied 74 and 6 patients with either isolated Cushing disease (CD) or GH‐ or PRL‐secreting PA, respectively. We also screened four pediatric patients with CD, and four with GH/PRL‐secreting tumors who had some syndromic features. There was one AIP mutation (p.Lys103Arg) among 74 CD patients. Two MEN1 mutations that occurred in patients with recurrent or difficult‐to‐treat disease were found among patients with CD. There was one MEN1 and three AIP mutations (p.Gln307ProfsX104, p.Pro114fsX, p.Lys241X) among pediatric patients with isolated GH‐ or PRL‐secreting PA and one additional MEN1 mutation in a patient with positive family history. There were no mutations in the PRKAR1A, CDKN1B, CDKN2C or GNAS genes. Thus, germline AIP or MEN1 gene mutations are frequent among pediatric patients with GH‐ or PRL‐secreting PA but are significantly rarer in pediatric CD; PRKAR1A mutations are not present in PA outside of Carney complex.     

An association between variants in the IGF2 gene and Beckwith-Wiedemann syndrome: interaction between genotype and epigenotype

Beckwith-Wiedemann syndrome (BWS) is a fetal overgrowth disorder involving the deregulation of a number of genes, including IGF2 and CDKN1C, in the imprinted gene cluster on chromosome 11p15.5. In sporadic BWS cases the majority of patients have epimutations in this region. Loss of imprinting of the IGF2 gene is frequently observed in BWS, as is reduced CDKN1C expression related to loss of maternal allele-specific methylation (LOM) of the differentially methylated region KvDMR1. The causes of epimutations are unknown, although recently an association with assisted reproductive technologies has been described. To date the only genetic mutations described in BWS are in the CDKN1C gene. In order to screen for other genetic predispositions to BWS, the conserved sequences between human and mouse differentially methylated regions (DMRs) of the IGF2 gene were analyzed for variants. Four single nucleotide polymorphisms (SNPs) were found in DMR0 (T123C, G358A, T382G and A402G) which occurred in three out of 16 possible haplotypes: TGTA, CATG and CAGA. DNA samples from a cohort of sporadic BWS patients and healthy controls were genotyped for the DMR0 SNPs. There was a significant increase in the frequency of the CAGA haplotype and a significant decrease in the frequency of the CATG haplotype in the patient cohort compared to controls. These associations were still significant in a BWS subgroup with KvDMR1 LOM, suggesting that the G allele at T382G SNP (CAGA haplotype) is associated with LOM at KvDMR1. This indicates either a genetic predisposition to LOM or interactions between genotype and epigenotype that impinge on the disease phenotype.     

Silver‐Russell patients showing a broad range of ICR1 and ICR2 hypomethylation in different tissues

Begemann M, Spengler S, Kanber D, Haake A, Baudis M, Leisten I, Binder G, Markus S, Rupprecht T, Segerer H, Fricke‐Otto S, Mühlenberg R, Siebert R, Buiting K, Eggermann T. Silver‐Russell patients showing a broad range of ICR1 and ICR2 hypomethylation in different tissues. In all known congenital imprinting disorders an association with aberrant methylation or mutations at specific loci was well established. However, several patients with transient neonatal diabetes mellitus (TNDM), Silver‐Russell syndrome (SRS) and Beckwith‐Wiedemann syndrome (BWS) exhibiting multilocus hypomethylation (MLH) have meanwhile been described. Whereas TNDM patients with MLH show clinical symptoms different from carriers with isolated 6q24 aberrations, MLH carriers diagnosed as BWS or SRS present only the syndrome‐specific features. Interestingly, SRS and BWS patients with nearly identical MLH patterns in leukocytes have been identified. We now report on the molecular findings in DNA in three SRS patients with hypomethylation of both 11p15 imprinted control regions (ICRs) in leukocytes. One patient was a monozygotic (MZ) twin, another was a triplet. While the hypomethylation affected both oppositely imprinted 11p15 ICRs in leukocytes, in buccal swab DNA only the ICR1 hypomethylation was visible in two of our patients. In the non‐affected MZ twin of one of these patients, aberrant methylation was also present in leukocytes but neither in buccal swab DNA nor in skin fibroblasts. Despite mutation screening of several factors involved in establishment and maintenance of methylation marks including ZFP57, MBD3, DNMT1 and DNMT3L the molecular clue for the ICR1/ICR2 hypomethylation in our patients remained unclear. Furthermore, the reason for the development of the specific SRS phenotype is not obvious. In conclusion, our data reflect the broad range of epimutations in SRS and illustrate that an extensive molecular and clinical characterization of patients is necessary.