Renal Abnormalities in Beckwith-Wiedemann Syndrome Are Associated with 11p15.5 Uniparental Disomy
Michael Goldman*,
Adam Smith,,
Cheryl Shuman||,
Oana Caluseriu,
Chihong Wei¶,
Leslie Steele¶,
Peter Ray,¶,#,
Paul Sadowski#,
Jeremy Squire**,
Rosanna Weksberg,,||,# and
Norman D. Rosenblum*,,
Divisions of *Nephrology and ||Clinical and Metabolic Genetics, Program in Developmental Biology, Research Institute, and ¶Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada; Institute of Medical Sciences, and Departments of #Molecular and Medical Genetics, Laboratory Medicine and Pathobiology, and Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; and **Ontario Cancer Institute, Toronto, Ontario, Canada.
Correspondence to Dr. Norman D. Rosenblum, Division of Nephrology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. Phone: 416-813-5667; Fax: 416-813-6271; E-mail: norman.rosenblum{at}sickkids.ca
ABSTRACT. Beckwith-Wiedemann syndrome (BWS) is a somatic overgrowthsyndrome characterized by a variable incidence of congenitalanomalies, including hemihyperplasia and renal malformations.BWS is associated with disruption of genomic imprinting and/ormutations in one or more genes encoded on 11p15.5, includingCDKN1C (p57KIP2). It was hypothesized that genotypic and epigenotypicabnormalities of the 11p15.5 region affecting CDKN1C were associatedwith renal abnormalities. Medical records for 159 individualswith BWS were reviewed. All underwent at least one abdominalultrasonographic evaluation. Testing for paternal uniparentaldisomy (UPD) at 11p15.5, CDKN1C mutations, and imprinting defectsat KvDMR1 was performed for 96, 32, and 47 patients, respectively.Of the 159 patients, 67 (42%) exhibited renal abnormalities,mainly nephromegaly (25%), collecting system abnormalities (11%),and renal cysts (10.5%). The frequency of renal lesions amongpatients who were tested for genetic abnormalities did not differfrom that among patients who were not tested. Paternal UPD wasdemonstrated in 22 of 96 cases (23%), CDKN1C mutations in eightof 32 cases (25%), and KvDMR1 imprinting defects in 21 of 47cases (45%). The 22 UPD-positive patients exhibited a significantlyhigher incidence of renal abnormalities (P = 0.0026). Surprisingly,the eight patients with CDKN1C mutations exhibited no significantincrease in the incidence of renal lesions (P = 0.29). Imprintingdefects at KvDMR1, which might downregulate CDKN1C, were alsonot associated with a significant difference in the incidenceof renal disease. Whereas UPD at 11p15.5 in BWS was associatedwith a higher incidence of renal abnormalities, mutations atCDKN1C and KvDMR1 imprinting defects were not, suggesting thatimprinted genes on 11p15.5 other than CDKN1C are critical forrenal development.
Beckwith-Wiedemann syndrome (BWS) is a congenital disorder characterizedby somatic overgrowth, macroglossia, anterior abdominal walldefects, and macrosomia. Additional but variable features includea typical facial appearance, ear lobe creases or pits, facialnevus flammeus, hemihyperplasia, neonatal hypoglycemia, organomegaly,genitourinary abnormalities, and a predisposition to developembryonal tumors (1,2). Renal abnormalities have been observedin up to 59% (range, 25 to 59%) of patients with BWS (2–4).The most common renal abnormalities are nephromegaly (19 to59%), simple cysts (13 to 19%), hydronephrosis (12 to 14%),and medullary cysts (5 to 13%). Other, less common findingsinclude nephrocalcinosis (with or without nephrolithiasis) (4%),increased cortical and pyramidal echogenicity (4%), a doublecollecting system (4%), and caliceal diverticula (1.3%). Themolecular mechanisms causing these renal abnormalities are undefined.
BWS is associated with disruption of genomic imprinting of oneor more genes in the 11p15.5 region. Genomic imprinting is aprocess that results in parent-of-origin-specific gene expression.Approximately 20% of sporadic BWS cases demonstrate mosaicismfor paternal uniparental disomy (UPD), with two paternally derivedcopies of chromosome 11p15.5 and no maternal contribution forthat region (5,6) (Figure 1). In approximately 50% of BWS cases,there is an epigenetic alteration at KvDMR1, which is a differentiallymethylated region at the 5'-end of an antisense transcript (KCNQ1OT1)that is not translated. In normal individuals, the KCNQ1OT1transcript is imprinted with monoallelic expression from thepaternally derived allele. The epigenetic alteration in 50%of BWS cases involves loss of maternal methylation at KvDMR1and biallelic expression of the antisense transcript KCNQ1OT1.KvDMR1/KCNQ1OT1 has been proposed as an imprinting control centerfor the proximal cluster of imprinted genes in the 11p15.5 region.These genes include CDKN1C (also known as p57KIP2), KCNQ1 (KVLQT1),SLC22A1L, and TSSC3. CDKN1C, a maternally expressed gene encodinga cyclin-dependent kinase inhibitor, is a negative regulatorof fetal growth. Mutations in this gene are detected in approximately40% of familial BWS cases and approximately 5% of nonfamilialcases (5,7). A second, more telomeric, imprinted gene clusteron chromosome 11p15.5 includes insulin-like growth factor 2(IGF2), which is a paternally expressed fetal growth factor,and H19, which is an untranslated RNA sequence. In 30% of BWScases, biallelic expression of IGF2 occurs in the presence ofnormal H19 expression and promoter methylation (8,9). Rarely,a primary methylation change at the H19 promoter, affectingH19 expression, occurs. In these cases, loss of maternal monoallelicexpression of H19 is associated with biallelic expression ofIGF2.
Figure 1. Map of imprinted genes on chromosome 11p15.5. Maternal (Mat) and paternal (Pat) contributions are indicated. The KvDMR1 region in intron 10 of KCNQ1 is represented by a gray box within the body of the KCNQ1 gene, and its allelic methylation is represented as CH3. Active transcription of an allele of an imprinted gene is represented by a white box with an arrow, whereas the silenced allele is indicated in black. The double hatch-marks represent several hundred kilobases of 11p15.5 that are not represented.
Investigation of the genetic features of BWS has begun to provideinsight into the molecular mechanisms that control the BWS phenotype.Positive correlations exist between UPD at 11p15.5 and hemihyperplasia(5) and tumors (10); between mutations in CDKN1C and omphalocele(7) and cleft palate (6); and between 11p15.5 duplication andneonatal hypoglycemia and developmental delay (11).
Studies in genetic murine models demonstrated that IGF2 andCDKN1C control growth and morphogenesis during kidney development(12,13). Although the renal phenotype in experimental murinemodels paralleling BWS gain-of-function IGF2 mutations demonstratedstriking nephromegaly and general overgrowth (14), loss-of-functionCDKN1C mutations produced cystic renal disease, as observedin BWS cases. Therefore, we hypothesized that CDKN1C is thegene responsible for the dysplastic/cystic renal disease componentof BWS. We predicted that the highest rate of renal abnormalitieswould be observed in cases with CDKN1C downregulation, including(1) CDKN1C mutations, (2) loss of KvDMR1 methylation (whichmight downregulate CDKN1C expression), and (3) 11p15 UPD (whichresults in a decrease in CDKN1C expression and an increase inIGF2 expression) (Figure 3). Therefore, we analyzed the renalphenotypes in these molecular subgroups of BWS (n = 159), including67 cases with renal abnormalities. Surprisingly, we observedthat renal abnormalities were associated with paternal 11p15.5UPD in BWS but not with CDKN1C mutations or KvDMR1 epigeneticlesions, suggesting that genes in the 11p15.5 region other thanCDKN1C are responsible for the renal anomalies in BWS.
Figure 3. Model of genomic imprinting on 11p15.5, including the imprinted genes CDKN1C and KCNQ1, containing the differentially methylated region KvDMR1 (gray box with CH3), a putative imprinting center. (A) Normal expression. Expression of CDKN1C (arrow) is preferentially from the maternal allele (95%), with slight expression from the paternal allele (5%) (23). (B) Paternal uniparental disomy (UPD). Patients with paternal UPD (two paternal copies for a segment of chromosome 11) demonstrate 10% of the normal expression of CDKN1C. However, 11p15.5 UPD extends to other imprinted genes and regulatory regions in band 11p15.5. (C) Mutation in the maternal CDKN1C allele. CDKN1C expression might be reduced to as little as 5%, the contribution from the imprinted paternal allele. (D) Imprinting defects in KvDMR1. Loss of methylation (LOM) has been postulated to downregulate expression of CDKN1C (24), as illustrated by the question mark.
Patients and Clinical Procedures
Medical records for 159 individuals with diagnoses of BWS werereviewed. Of these, 72 were male and 87 were female. The medianage at the time of the last clinical review was 6.5 yr (range,13 to 39 mo). Seventy-six patients were monitored at the Hospitalfor Sick Children (HSC) (Toronto, Ontario, Canada), and 83 werereferred from other centers in North America. The clinical diagnosisof BWS was based on the presence of at least three of the followingfeatures (or at least two of these features plus one of neonatalhypoglycemia, abdominal organomegaly, or renal malformation):macrosomia, macroglossia, hemihyperplasia, ear creases/pits,abdominal wall defects (omphalocele or umbilical hernia), orembryonal tumors.
All patients monitored at HSC were offered abdominal computedtomographic or magnetic resonance imaging assessments at theage of 6 mo or at the time of diagnosis (if patients presentedat >6 mo of age). All patients underwent abdominal ultrasonographyevery 3 mo until 8 yr of age. If patients with BWS presentedat >8 yr of age, they underwent at least one ultrasonographicevaluation. Further imaging investigations, including renalscanning, voiding cystourethrography, and excretory urography,were performed if clinically indicated. For patients referredfrom institutions other than HSC, data from at least one ultrasonographicevaluation were available.
DNA or RNA was obtained for analyses of chromosome 11 UPD, CDKN1Cmutations, and KvDMR1 methylation status. UPD testing resultsare reported for the 96 cases (48 from HSC and 48 from othercenters) for which a recently developed, highly informative,sensitive test was applied (15). CDKN1C testing was performedfor patients with a family history of BWS and/or clinical featuressuggesting a CDKN1C mutation (e.g., omphalocele or cleft palate)(16). Testing for KvDMR1 methylation status, with a relativelyrecently developed test, was performed for recently recruitedpatients. Of the 96 patients tested for UPD, 21 were testedfor CDKN1C mutations and 34 were tested for KvDMR1 methylationstatus. All three tests were performed for 17 patients.
A subset of the patients described here were described elsewhere(15) in an analysis of tumor predisposition. These studies wereapproved by the Research Ethics Board of the HSC.
Cell Cultures
Lymphoblast lines were maintained in RPM1 1640 medium supplementedwith 15% fetal calf serum. Fibroblast strains were maintainedfor <10 passages in -minimal essential medium supplementedwith 10% fetal calf serum.
Chromosome 11 UPD Analysis
Chromosome 11 UPD analysis was performed as described previously(15). Briefly, genomic DNA was extracted from either peripheralblood, cultured lymphoblasts, or skin fibroblasts from the probandsand their parents. Quantitative multiplex-PCR, using highlypolymorphic short tandem repeat (STR) markers, was performedwith three markers within (TH, D11S2362, and D11S1997) and twomarkers distal to (D11S1998 and D11S1974) the BWS critical regionat 11p15.5, to detect somatic cell rearrangements giving riseto paternal UPD of the chromosome 11p15.5 region. The percentageof paternal UPD of alleles at 11p15.5 in the probands was determinedfrom informative alleles for at least two DNA markers withinthe BWS critical region that exhibited dosage increases of >20%,as determined with the following calculation: (peak area ofpaternal allele - peak area of maternal allele)/(peak area ofpaternal allele + peak area of maternal allele). Allele dosageswere measured because mosaicism for 11p15.5 UPD exists amongthese patients.
Southern Blot Analysis of KvDMR1
For analysis of KvDMR1 methylation, genomic DNA was digestedwith EcoRI and NotI. Digestion products were subjected to electrophoresison 0.8% agarose gels and were then transferred to GeneScreenPlus membranes (NEN, Boston, MA). Blots were hybridized witha 400-bp, [-32P]dCTP-labeled, DMR probe (a kind gift from M.Higgins Roswell, Park Cancer Institute, Buffalo, NY) (17). Themethylation index was determined by dividing the OD of the 4.2-kbband by the combined OD values of the 4.2- and 2.7-kb bands(17).
CDKN1C Mutation Screening CDKN1C mutation screening was performed as described by Li etal. (16).
Statistical Analyses
Genotype-phenotype correlations between 11p15.5 UPD, CDKN1Cmutations, KvDMR1 methylation, and renal abnormalities wereassessed by using the 2 test. P values of <0.05 were consideredstatistically significant.
Renal Abnormalities among Patients with BWS Nonmalignant Lesions.
Nonmalignant renal abnormalities occur with an incidence ofup to 59% among patients with BWS. Nephromegaly accounts forapproximately one-half of these lesions (2–4). Of our159 patients with BWS, 67 (42%) exhibited renal lesions (Table 1).Nephromegaly was the most common finding (25%), followedby abnormalities of the collecting system (renal calyces, pelvis,and ureters) (11%). Hydronephrosis was present in 10 of 17 patientswith collecting system abnormalities. For six of those patients,the cause was not defined, despite imaging of the kidney andurinary tract. For another three, the hydronephrosis was mildand the cause was not investigated. Yet another patient exhibitedmoderate hydronephrosis and hydroureter secondary to vesicoureteralreflux. Interestingly, severe vesicoureteral reflux (grade 4to 5) was detected for one patient for whom hydronephrosis wasnot detected by ultrasonography. Four patients exhibited calicealdiverticula, as detected by ultrasonography and confirmed bycomputed tomography. Three patients exhibited a double collectingsystem. Simple cortical cysts were detected for five (3%) andmultiple medullary cysts for 12 (7.5%) of our patients. Nephrocalcinosisand/or nephrolithiasis was observed for six patients (4%) inthis study. Increased cortical echogenicity was observed fortwo patients (1%). Taken together, our results demonstratednonmalignant renal abnormalities in 42% of our cases. The mostcommon abnormality was nephromegaly (25%), followed by collectingsystem abnormalities (11%).
Table 1. Renal abnormalities among patients with Beckwith-Wiedemann syndrome
Malignant Lesions and Hemihyperplasia.
Estimates of the risk of tumor development among patients withBWS vary widely (4 to 21%) (1,18,19). In this series, tumorswere diagnosed for 22 of 159 patients (14%), including hepatoblastomasin seven cases, adrenal carcinoma in one, rhabdomyosarcoma inone, and Wilms’ tumor in 13 (8% of all patients). Therisk of Wilms’ tumor in BWS is significantly increasedamong patients with hemihyperplasia and/or nephromegaly (1,18,20).Previous studies demonstrated that hemihyperplasia occurs inapproximately 25% of BWS cases (2). In this study, hemihyperplasiawas detected for 53 of 159 patients (33%) and was more frequentamong patients with tumors (11 of 22 patients), compared withthose without tumors (43 of 137 patients) (P = 0.02). Therefore,patients in this series demonstrated a 14% incidence of tumorformation and a positive association between hemihyperplasiaand tumor formation, consistent with previous reports.
Genetic Abnormalities among Patients with BWS
Several regional or single-gene alterations have been demonstratedamong patients with BWS (6). These abnormalities encompass regionsof 11p15.5 that encode genes including IGF2 and CDKN1C. Becauseloss-of-function CDKN1C mutations in mice affect renal morphogenesis(12,13), we hypothesized that genotypic and epigenotypic abnormalitiesof the 11p15.5 region affecting CDKN1C might be associated withrenal abnormalities. To investigate this possibility, we firstidentified imprinting abnormalities and CDKN1C mutations amongpatients for whom biologic samples were available. To determinewhether the subsets of patients for whom genetic testing wasperformed were representative of the total population, we comparedphenotypes between the groups. Our results demonstrated thatthe incidences of renal lesions, hemihyperplasia, and tumorswere similar among patients who were tested for UPD and thosewho were not (tested for UPD versus not tested: renal lesions,P = 0.402; hemihyperplasia, P = 0.302; tumors, P = 0.735). Similarly,we could not detect differences in these abnormalities betweenindividuals who were tested and those were not tested for mutationsin CDKN1C (tested for CDKN1Cversus not tested: renal lesions,P = 0.847; hemihyperplasia, P = 0.485; tumors, P = 0.805) ormethylation at KvDMR1/KCNQ1OT1 (tested for methylation at KvDMR1/KCNQ1OT1versus not tested: renal lesions, P = 0.260; hemihyperplasia,P = 0.902; tumors, P = 0.450). These data demonstrated thatthe subsets of patients with BWS who were tested for 11p15 UPD,methylation at KvDMR1, or CDKN1C mutations were representativeof our BWS population.
Imprinting Abnormalities
We tested for imprinting abnormalities by determining the methylationstatus of KvDMR1 and the presence of paternal 11p15 UPD. Whereasonly the paternal allele of KCNQ1OT1 is expressed in normalindividuals, in association with methylation of the maternalKvDMR1 allele (Figure 1), there is biallelic expression of KCNQ1OT1in approximately 50% of patients with BWS, in association withthe loss of maternal methylation at KvDMR1. We tested 47 patientsfor methylation at the KvDMR1 locus and observed loss of maternalmethylation in 21 of 47 non-UPD cases (45%), consistent withprevious reports (Figure 2B). Paternal UPD at 11p15.5 is observedin approximately 20% of BWS cases (6). We tested 96 patientsfor 11p15.5 UPD, using quantitative multiplex-PCR of highlypolymorphic STR markers within the BWS critical region. We detectedUPD in 22 of 96 cases (23%), consistent with previous reports(Figure 2C).
Figure 2. (A) CDKN1C mutation analysis. (Left) Normal sequence for nucleotides 426 to 438 in exon 2 of CDKN1C. (Right) Heterozygous CT mutation at nucleotide 432 (numbered relative to the published cDNA sequence; Genbank Accession No. 4557440), resulting in a single amino acid substitution of a glutamine to leucine at residue 58 of the 316-amino acid protein. (B) DNA methylation analysis of KvDMR1. Southern blots of DNA samples digested with NotI and EcoRI hybridized with a [32P]dCTP-labeled DMR probe are presented. The upper band (4.2 kb) is of maternal origin and the lower band (2.7 kb) of paternal origin. Blots indicate a control subject with a methylation index of 0.50 (left), a patient with Beckwith-Wiedemann syndrome (BWS) with a methylation index of 0.24 (center), and a patient with BWS with a methylation index of 0 (right). LOM, loss of methylation. (C) Quantitative multiplex-short tandem repeat analysis to detect paternal chromosome 11 UPD. In a child with BWS and paternal UPD11, an increase in dosage of the paternally derived allele, compared with the maternally derived allele, is observed at DNA markers within the 11p15.5 region [D11S1997 (blue)], D11S2362 (green), and TH (black)]. This is in contrast to the normal dosage of two DNA markers on the q arm of chromosome 11 [D11S1974 (green) and D11S1998 (black)].
CDKN1C Mutations CDKN1C (p57KIP2), which encodes a cyclin-dependent kinase inhibitor,is maternally expressed in normal individuals. Because mutationsin CDKN1C occur in both familial and nonfamilial BWS cases andhomozygous mutational inactivation of murine CDKN1C causes renaldysplasia, we tested for mutations by sequencing the entirecoding region (Figure 2A). Individuals with a positive familyhistory were first screened for CDKN1C mutations. Mutationswere detected for eight of 32 patients. Of these eight patients,three were derived from a single family and carried the samemutation; therefore, the rate of mutation in our familial caseswas 20%, consistent with previously published data (16).
Genotype-Phenotype Correlations
Characterization of the clinical features and genetic/epigeneticabnormalities of a representative subset of patients with BWSprovided the basis for determining whether downregulation ofCDKN1C, via mutation or imprinting defects (such as 11p15.5UPD or loss of methylation at KvDMR1), was positively associatedwith the occurrence of renal abnormalities (Table 2). Of interest,11p15 UPD-positive patients demonstrated a significantly higherincidence of renal abnormalities (P = 0.0026). Patients withBWS and mosaicism for 11p15 UPD also demonstrated significantlyhigher incidences of hemihyperplasia and tumors, compared with11p15 UPD-negative cases, as previously reported (5,10). Todetermine the contributions of hemihyperplasia and tumors tothe new renal association demonstrated by our data, we performeda subgroup analysis of patients with renal lesions but withouthemihyperplasia or tumors. For these patients, a positive statisticalcorrelation was demonstrated between 11p15 UPD and renal lesions(P = 0.017 and P = 0.032, respectively). This positive correlationpersisted when we controlled for the presence of nephromegaly(P = 0.003) or both nephromegaly and Wilms’ tumor (P =0.003). Surprisingly, we could observe no statistically significantcorrelations between CDKN1C mutations and renal lesions or betweenhemihyperplasia and tumors, although there was a tendency towardlower incidences of these abnormalities among patients withCDKN1C mutations (Table 2). Similarly, we could not demonstratesignificant correlations between the incidences of renal lesions,hemihyperplasia, or tumors (mostly Wilms’ tumors) andabnormal imprinting at the KvDMR1 locus (P = 0.181, P = 0.60,and P = 0.99, respectively).
Table 2. Genotype (UPD and CDKN1C)-phenotype (renal lesions, hemihyperplasia, and tumor) analysisa
Our results confirm previously reported results suggesting positivecorrelations between UPD and hemihyperplasia and tumor formation.More importantly, our data provide the first demonstration thatrenal lesions in BWS are positively associated with paternalUPD at 11p15.5 but not with CDKN1C mutations or imprinting defectsat KvDMR1.
This study represents, to our knowledge, the first analysisof correlations between renal abnormalities and molecular defectsamong patients with BWS. On the basis of data obtained withmice (12), we hypothesized that downregulation of CDKN1C wouldbe associated with renal malformations among patients with BWS.Consistent with our hypothesis, we expected that renal malformationswould be positively associated with CDKN1C mutations in BWS,as well as with two imprinting defects that decrease CDKN1Cactivity, i.e., (1) paternal UPD, in which CDKN1C activity isreduced because of the presence of two paternally derived CDKN1Calleles, and (2) KvDMR1 imprinting defects, which might causedownregulation of CDKN1C. Therefore, we investigated genotype-phenotypecorrelations for renal abnormalities among patients with BWSand CDKN1C mutations, 11p15.5 UPD, and KvDMR1 loss of imprinting.Of the 159 patients in this study, 67 (42%) exhibited a varietyof renal abnormalities (Table 1). The 11p15 UPD-positive patientsexhibited a significantly higher incidence of renal abnormalities,in contrast to patients with CDKN1C mutations or KvDMR1 lossof imprinting (Table 2). Our results demonstrated a novel associationbetween 11p15.5 UPD and renal lesions among patients with BWS.
In BWS, paternal 11p15.5 UPD is observed in approximately 20%of cases and abnormal KvDMR1 methylation in approximately 50%of cases (5–7). Our findings of 23% of cases with paternal11p15.5 UPD and 45% with abnormal KvDMR1 methylation are consistentwith these results. Five percent of sporadic cases and 40% offamilial cases have been reported to involve CDKN1C germlinemutations (5–7). We observed CDKN1C mutations for eightof 32 patients (25%) who were tested. Three of the patientswere from a single family, and five were from different families.The relatively high incidence is explained by the fact thata group of individuals with positive family histories was includedin the study. Although not all of our patients underwent moleculartesting, we determined that the incidences of renal lesions,hemihyperplasia, and tumors were similar for patients who weretested for UPD, CDKN1C mutations, and KvDMR1 methylation andthose who were not tested.
Abnormalities of the renal collecting system that are associatedwith BWS include hydronephrosis and/or hydroureter, vesicoureteralreflux, double collecting system, and caliceal diverticula.These abnormalities have been observed in approximately 14%of BWS cases (2,3). Consistent with those reports, 17 of thepatients (11%) in our study demonstrated such lesions. The mostprevalent abnormality was hydronephrosis. Although determinationof the underlying cause was limited by the retrospective natureof our study, imaging results for the patients with the mostsevere grades of hydronephrosis demonstrated the absence ofany fixed obstructing lesion. Therefore, it is likely that,in most cases, dilation of the collecting system was secondaryto nonobstructive abnormalities, perhaps including abnormalitiesof the pelvic and ureteric smooth muscle layers. Less commonabnormalities of the collecting system included caliceal diverticulaand vesicoureteral reflux. The similarity of the prevalenceof those abnormalities to that observed in the general population(3) (2.5 and 1.2%, respectively) suggested that the presenceof those lesions was coincidental and not secondary to BWS.
Cysts represented the most prevalent renal abnormality amongour patients. The majority of the cysts were located in themedulla, rather than the cortex. This is in contrast to findingsfor patients without BWS, for whom isolated cysts are rare andare located in the cortex (21). Medullary cysts are derivedfrom dilated collecting ducts and loops of Henle and are formedduring development of the renal collecting system and medulla(22). Therefore, these cysts may represent a more severe phenotypein a continuum of collecting system abnormalities, with mildpelvic dilation representing a mild phenotype. The relativelyhigh prevalences of nephrocalcinosis and nephrolithiasis inthis study (4%) and other studies (3,4) might be secondary tothe congenital abnormalities of the collecting system amongthe patients. Two of the six affected patients in our studyexhibited medullary cysts associated with nephrocalcinosis,two exhibited hydronephrosis (which might predispose them tothe development of renal stones), and two exhibited isolatednephrocalcinosis. Laboratory data (urinary calcium, creatinine,oxalate, amino acid, uric acid, and citrate levels) were availablefor four patients. Three of the patients demonstrated hypercalciuria,and results were normal for the fourth. Taken together, theseabnormalities highlight the pathogenic role of collecting systemmaldevelopment in BWS and suggest the need for future studiesaimed at defining the natural history of these abnormalities.
Our demonstration of a novel genotype-phenotype correlationbetween paternal 11p15.5 UPD and renal lesions suggests thatthis genomic region consists of genes that control renal morphogenesis.Disruption of the function of these genes might cause abnormalpatterning of the collecting system in the cortex and medulla,leading to the development of renal cysts and collecting systemabnormalities. The 11p15.5 region associated with BWS spans1 Mb containing several imprinted genes, including IGF2, H19,CDKN1C, and KCNQ1/KCNQ1OT1 (Figure 1). Both CDKN1C and IGF2were demonstrated to participate in the control of renal morphogenesisin animal studies; however, overexpression of IGF2 producesnephromegaly in mice but does not produce renal medullary dysplasia,as observed for some patients with BWS (14). In contrast, p57KIP2-(-/-)mice do exhibit such renal medullary dysplasia (12). Mutationsin H19 cause enhanced expression of IGF2, and mice with mutationsof both p57KIP2 and H19 exhibit elevated serum IGF-2 levelsand more severe renal dysplasia, compared with p57KIP2-(-/-)mice (13). That is, increased IGF2 expression exacerbates thep57KIP2-(-/-) renal phenotype in mice but is not a primary causeof that phenotype. Our results demonstrating that CDKN1C mutationsand imprinting defects at KvDMR1 were not correlated with renalabnormalities strongly suggest that defects in imprinted genesin the 11p15 region other than CDKN1C produce renal abnormalitiesamong human subjects with BWS. Whereas both primary CDKN1C mutationsand 11p15.5 UPD significantly reduced CDKN1C expression, anincrease in renal malformations was noted only with 11p15.5UPD (Figure 3, B versus C). Similarly to primary CDKN1C mutations,loss of methylation at KvDMR1 was not associated with renalmalformations (Figure 3D). Therefore, imprinted genes or regulatoryelements other than CDKN1C or KvDMR1 must account for renaldysplasia among human subjects with BWS. Future experimentsdirected at determining the role of imprinted genes in thisregion hold the promise of providing new insights into the geneticcontrol of renal development and, more generally, the relationshipbetween the control of growth and morphogenesis.
Acknowledgments
We thank all of the family members who participated in thisstudy. The KvDMR1 probe was generously provided by Dr. M. Higgins.This research was supported by the National Cancer Instituteof Canada, with funds from the Canadian Cancer Society (to Dr.Weksberg) and the Canadian Institutes of Health Research (toDr. Rosenblum). We acknowledge the assistance of Ingrid Ambus,who was the recipient of a Starbucks Clinical Genetics ResearchStudentship Award.
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Received for publication March 6, 2002.
Accepted for publication April 19, 2002.
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R H Scott, C A Stiller, L Walker, and N Rahman Syndromes and constitutional chromosomal abnormalities associated with Wilms tumour
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Abstract
Introduction
-minimal essential medium supplemented with 10% fetal calf serum. 
2 test. P values of <0.05 were considered statistically significant. 
T mutation at nucleotide 432 (numbered relative to the published cDNA sequence; Genbank Accession No. 4557440), resulting in a single amino acid substitution of a glutamine to leucine at residue 58 of the 316-amino acid protein. (B) DNA methylation analysis of KvDMR1. Southern blots of DNA samples digested with NotI and EcoRI hybridized with a [32P]dCTP-labeled DMR probe are presented. The upper band (4.2 kb) is of maternal origin and the lower band (2.7 kb) of paternal origin. Blots indicate a control subject with a methylation index of 0.50 (left), a patient with Beckwith-Wiedemann syndrome (BWS) with a methylation index of 0.24 (center), and a patient with BWS with a methylation index of 0 (right). LOM, loss of methylation. (C) Quantitative multiplex-short tandem repeat analysis to detect paternal chromosome 11 UPD. In a child with BWS and paternal UPD11, an increase in dosage of the paternally derived allele, compared with the maternally derived allele, is observed at DNA markers within the 11p15.5 region [D11S1997 (blue)], D11S2362 (green), and TH (black)]. This is in contrast to the normal dosage of two DNA markers on the q arm of chromosome 11 [D11S1974 (green) and D11S1998 (black)].