American Journal of Medical Genetics Part C (Semin. Med. Genet.) 137C:4 –11 (2005) A R T I C L E Clinical and Molecular Overlap in Overgrowth Syndromes GENEVIÈVE BAUJAT, MARLÈNE RIO, SYLVIE ROSSIGNOL, DAMIEN SANLAVILLE, STANISLAS LYONNET, MARTINE LE MERRER, ARNOLD MUNNICH, CHRISTINE GICQUEL, LAURENCE COLLEAUX, AND VALÉRIE CORMIER-DAIRE* Here, we report the clinical and molecular analysis of 75 patients with overgrowth and mental retardation, including 45 previously reported cases [Rio et al., 2003; Baujat et al., 2004]. Two groups are distinguished: group I corresponding to patients with recognizable overgrowth syndromes (Sotos syndrome (SS), Weaver syndrome (WS), Beckwith–Wiedemann syndrome, Simpson–Golabi–Behmel syndrome (SGBS), and del(22)(qter) syndrome) (60 cases) and group II corresponding to unclassified cases (15 patients). We investigated NSD1 and GPC3 deletions or mutations, 11p15 abnormalities, and 22qter deletions. Surprisingly, in Group I, two SS patients had 11p15 abnormalities and two patients with Beckwith–Wiedemann syndrome had NSD1 aberrations. In group II, two cases of del(22)(qter) were identified but neither NSD1, 11p15, nor GPC3 abnormalities were detected. These results emphasize the clinical and molecular overlap in overgrowth conditions. ß 2005 Wiley-Liss, Inc. KEY WORDS: Sotos syndrome; Weaver syndrome; Beckwith–Wiedemann syndrome; Simpson–Golabi–Behmel syndrome; del(22)(qter) INTRODUCTION Although problems in overgrowth were documented and appreciated in the nineteenth century, conditions such as Beckwith–Wiedemann syndrome (BWS, OMIM 130650) and Sotos syndrome Dr. Geneviève Baujat has a Fellowship in Pediatrics and received her M.D. in Genetics from the University R. Descartes in Paris. She currently focuses on developmental pediatrics and overgrowth syndromes. Valérie Cormier-Daire M.D., Ph.D., is a Medical Geneticist in the Department of Medical Genetics of Necker Enfants Malades Hospital in Paris, France. She has first trained in Pediatrics and then in Medical Genetics. Her PhD was focused on mitochondrial disorders. She has spent one year in Los Angeles at the Skeletal Dysplasia Registry. She is now involved at a clinical and research level in the skeletal dysplasia field and has a particular interest in syndromes and overgrowth conditions. She is the coauthor of more than 140 articles in the medical and scientific literature. Grant sponsor: Fondation pour la Recherche Medicale (FRM); Grant sponsor: le Fond de Recherche de l’Assistance Publique des Hôpitaux de Paris (FR-APHP). *Correspondence to: Valérie CormierDaire, M.D., Ph.D., Département de Génétique Médicale, Hôpital Necker-Enfants Malades, 149 rue de Sèvres, 75743 Paris cedex 15, France. E-mail: firstname.lastname@example.org DOI 10.1002/ajmg.c.30060 ß 2005 Wiley-Liss, Inc. (SS, OMIM 117550) were delineated by the 1960s. Other distinct overgrowth syndromes have been defined since in the early 1980s, such as Weaver syndrome (WS, OMIM 277590) , Perlman syndrome (PS, OMIM 267000) , Simpson–Golabi–Behmel syndrome (SGBS, OMIM 312870) , and Bannayan–Riley–Ruvalcaba syndrome (OMIM 153480) . This clinical overlap makes diagnosis of overgrowth syndromes often difficult [Verloes et al., 1995; Opitz et al., 1998]. More recently, molecular overlap has been demonstrated within this group of childhood overgrowth conditions [Li This clinical overlap makes diagnosis of overgrowth syndromes often difficult. More recently, molecular overlap has been demonstrated within this group of childhood overgrowth conditions. et al., 2001; Baujat et al., 2004]. Finally, large numbers of patients with overgrowth syndromes do not belong to any recognizable condition. Here, we report the clinical and molecular analysis of a series of 75 patients with overgrowth and mental retardation syndromes and provide additional evidence of clinical and molecular overlap in these conditions. MATERIALS AND METHODS Patients High-resolution chromosomal analysis and molecular analysis for fragile X syndrome were systematically performed before inclusion of the patients. Three unbalanced rearrangements were identified in patients with overgrowth and mental retardation: one case of mosaicism for 20p11.2 trisomy, involving a region that contains the somatosatin receptor 4 gene (SSTR4); one child with an overgrowth syndrome resembling SS [Faivre et al., 2000]; a 6q16;13q14 translocation, resulting in 6q16 deletion; and, two sibs with 15q26.1-qter trisomy involving a region that contains IGF1 [Faivre et al., 2002]. These three patients were therefore excluded from this study. ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) Seventy-five patients were included in the series. Among them, 45 have been previously reported [Rio et al., 2003; Baujat et al., 2004]. They all had the following manifestations: height > 97th centile, OFC > 97th centile, developmental delay, and at least two minor features among the following: bone age >90th centile, abnormal craniofacial features, and congenital malformations. All patients were regularly followed in the Department of Medical Genetics of Necker–Enfants Malades hospital for 10 years and phenotypically scored by clinical geneticists. They were classified into two groups: group I (60 cases) included patients with a recognizable overgrowth disorder (38 SS, 6 WS, and 2 SGBS). Fourteen patients with BWS with some degree of mental retardation were also included in this group. Group II (15 cases) included patients with unclassified overgrowth syndromes. The rationale for the molecular analysis of these patients was: Group I patients were first investigated for suspected molecular defects: NSD1 (nuclear receptor SET domain containing protein) screening in patients with SS and WS, 11p15 region in BWS, and GPC3 screening in SGBS. When no abnormality was found, patients were tested for other molecular aberrations (Fig. 1). Group II patients were screened for NSD1 gene, 11p15 region, GPC3, and 22qter region. Cytogenetic and Molecular Studies Consent was obtained from all patients and/or parents. Genomic DNA from each patient was isolated from blood lymphocytes using a Nucleon kit (Amersham, UK) according to the manufacturer’s instructions. NSD1 Analysis To identify microdeletions in NSD1, patients and their parents were genotyped using four polymorphic microsatellite markers (two intragenic and two located next to the NSD1 gene), according to Rio et al. . Patients who were homozygous for both intragenic markers were subsequently screened for a whole gene deletion by FISH analysis using the PAC RPCI-1 118M12 as a probe. When NSD1 microdeletion was excluded by genotyping and FISH, patients were then 5 selected for NSD1 sequencing according to Rio et al. . 11p15 Investigation To screen for deletions or uniparental disomy (UPD) of the 11p15 region, three polymorphic microsatellite markers (D11S4046, D11S1338, and D11S1346) were genotyped in patients and their parents. Deletion and disomy were further confirmed by performing FISH analysis, using PACs RP11896B12 and RP11-908H22 probes, located on distal chromosome 11p. The methylation status of KCNQ1OT1 and H19 in the 11p15 region was subsequently investigated, as described by Gaston et al. . Finally, a germ line CDKN1C mutation was searched for by direct sequencing [Gaston et al., 2000]. GPC3 Mutation Analysis Direct sequencing of the GPC3 gene was performed, using primers as previously reported [Huber et al., 1997]. del(22)(q13) Detection Patients and their parents were genotyped using three polymorphic micro- Figure 1. Rationale for the molecular analysis of group I. 6 AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) ARTICLE TABLE I. Primer Sequences for Chromosome 22qter Microsatellite Markers Microsatellite DNA marker D22S1169 Z821 89 AL022 327 Primer sequence (50 !30 ) Distance from the p telomere Forward Reverse Approximate size (bp) 47.63 48. 48.6 GCACACACATGCACATAATC AATTCCCACCAGGATGCT AGAGGGAGAGTCTCTTTCTC AACAAAACTTCCAGCAGACG ATTTTGCGGGCAGGATTCAG GACATTGCCTCTTAGTTCAC 124 284 166 and 7968delGACA) [Baujat et al., 2004]. In these four cases, retrospective analysis supported the initial diagnosis (Fig. 2A,B, Table IIIA,B). Finally, within group I, no molecular basis was identified in 24 patients (11 SS, 3 WS, and 10 BWS). In group II (15 patients), the systematic molecular analysis permitted identification of two cases of del(22) (qter) (Fig. 3). No NSD1, 11p15, or GPC3 abnormalities were found. Finally, within this group II, the molecular basis of 13 patients remained unidentified. satellite markers of the 22qter region, located near SHANK3 (Table I). Fluorescent genotyping was performed as previously described [Rio et al., 2002]. The deletion was then confirmed by FISH analysis using the cosmid 106G1220P [Bonaglia et al., 2001]. RESULTS Table II summarizes the results. In group I, 25/38 SS patients, and 3/6 WS patients had an NSD1 abnormality (8 deletions and 20 intragenic mutations, 2/14 patients with BWS had an 11p15 abnormality (1 CDKN1C mutation, 514delAC, and 1 KCNQ1OT1 demethylation), and 2/2 patients with SGBS had GPC3 mutations (1562C!T and 749G!A). The secondary molecular analysis in the remaining patients of this group (n ¼ 28) allows the identification of four additional aberrations: (1) 11p15 paternal isodisomy in one case and partial demethylation of KCNQ1OT1 with normal methylation of the H19 in a second case of SS; and (2) NSD1 mutations in two cases of BWS (4976insG Patients with specific clinical recognized entities (group I) were the most frequent in our series (60/75 patients) and, among them, 53% were confirmed by molecular diagnosis. SS was the most common condition in our series and may be the most often diagnosed overgrowth syndrome. DISCUSSION The series of 75 patients reported here represents nosologic difficulties faced by clinical geneticists attempting to diagnose, molecularly diagnose, and counsel in overgrowth conditions. Table IV lists the classical findings in the five major overgrowth disorders with known molecular abnormalities. Patients with specific clinical recognized entities (group I) were the most frequent in our series (60/75 patients) and, among them, 53 % were confirmed by molecular diagnosis (Table II). SS was the most common condition in our series (38/60) and may be the most often diagnosed overgrowth syndrome. WS, now described as allelic to SS, is less frequently observed (six cases in our series). NSD1 abnormalities were found in 65% of our SS cases and in 50% of TABLE II. Results of the Molecular Screening Molecular defects Clinical diagnosis Group 1: Sotos syndrome Weaver syndrome Beckwith–Wiedemann syndrome Simpson–Golabi–Behmel syndrome del(22)(qter) syndrome Total Group 2: unclassified Total Number of patients NSDI 11p15 GPC3 del (22)(qter) No molecular basis 38 6 14 2 0 60 15 75 25 3 2 0 0 30 0 30 2 0 2 0 0 4 0 4 0 0 0 2 0 2 0 2 0 0 0 0 0 0 2 2 11 3 10 0 0 24 13 37 ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) TABLE IIIA. Patients Clinically Diagnosed as Beckwith–Wiedemann Syndrome with NSD1 Mutations (n ¼ 2)a Wiedemann syndrome features of Sotos syndrome Features of Beckwith Presence of Postnatal overgrowth Hypoglycemia Abdominal wall defect Hemihypertrophy Genito-urinary abnormalities (renal cysts, ureteral reflux) Absence of Macroglossia Ear lobe crease Frontal angioma Visceromegaly a Presence of Postnatal overgrowth Mental retardation Scoliosis 2 1 2 1 2 1 2 (mild) 1 Absence of Advanced bone age Sotos facial features CNS malformations Seizures Atypical feature: craniosynostosis in one case. our WS cases, and this is consistent with previously published series [Douglas et al., 2003; Turkmen et al., 2003]. Two children with typical manifestations of SGBS had GPC3 mutations. Finally, 14 patients with BWS in our series all had some degree of mental deficiency, which is not usually part of the BWS spectrum, perhaps explaining the low percentage of 11p15 abnormalities in these patients. These findings illustrate the importance of clinical analysis in overgrowth conditions. When a diagnosis is made by physical examination and confirmed by molecular testing, clinical and molecular overlap sometimes occurs (Fig. 4). Clinical similarities between SS and WS and between BWS, SGBS, and PS have been reported elsewhere [Verloes et al., 1995; TABLE IIIB. Patients Clinically Diagnosed as Sotos Syndrome (SS) With 11p15 Abnormalities (n ¼ 2)a Features of Beckwith– Wiedemann syndrome Features of Sotos syndrome Presence of Prenatal overgrowth Postnatal overgrowth Macrocephaly Sotos facial features Mental retardation Seizures Scoliosis Absence of CNS malformations a 1 2 2 2 2 1 1 (severe) Atypical feature: arachnodactyly in one case. Presence of Prenatal overgrowth Postnatal overgrowth Congenital heart defect Septal hypertrophy Absence of Macroglossia Ear lobe crease Frontal angioma Hypoglycemia Abdominal wall defect Visceromegaly Hemihypertrophy 1 2 1 1 7 Opitz et al., 1998; Li et al., 2001]. SS and WS share some features in common, although distinctive features are present in WS (hoarse low-pitched cry, distinctive facial appearance, deep-set nails, finger pads, camptodactyly, and metaphyseal flaring) [Opitz et al., 1998]. The identification of NSD1 aberrations in both syndromes demonstrates that the two conditions are allelic. Several patients have been reassessed and diagnosed as having SGBS after an initial diagnosis of BWS [Verloes et al., 1995; Li et al., 2001]. Indeed, both conditions share many features, particularly during the neonatal period: fetal overgrowth, macroglossia, hernias, visceromegaly, hypotonia, and hypoglycemia. The molecular bases of these conditions can explain the clinical overlap. BWS is caused by dysregulation in the expression of imprinted genes in the 11p15 region, resulting in overexpression of IGF2. SGBS is caused by mutations and deletions in GPC3 and this gene encodes an extracellular proteoglycan, glypican 3, which plays an important role in the growth control of embryonic mesodermal tissue. Glypican 3 can interact with IGF2 and forms a complex that modulates IGF2 action. Therefore, a defect in glypican 3 should result in increased IGF2. PS is a rare fetal overgrowth disorder with nephroblastomatosis, distended abdomen, and hypoglycemia. PS differs from BWS by the absence of macroglossia, omphalocele and ear creases, and by the presence of macrocephaly, deep-set eyes, broad and low nasal bridge, long everted upper lip, and low-set ears. However, the observation of 11p15 aberrations in some PS cases suggests that PS belongs to the phenotypic spectrum of BWS [Verloes et al., 1995]. More surprisingly, our molecular findings in clinically recognized conditions illustrate overlap between BWS and SS [Baujat et al., 2004]. Two patients clinically diagnosed as BWS had NSD1 mutations (Table IIIA, Fig. 2A). In both cases, the diagnosis of BWS was based on macrosomia, abdominal wall defect, genitourinary anomalies, hemihyperplasia (1/2), neonatal hypoglycemia (1/ 2), and absence of advanced bone age. 8 AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) ARTICLE More surprisingly, our molecular findings in clinically recognized conditions illustrate overlap between BWS and SS. Two patients clinically diagnosed as BWS had NSD1 mutations. Figure 2. A: Facial features of a patient with Beckwith–Wiedemann syndrome at 5 years of age with an NSD1 mutation. Note the absence of frontal bossing, hypertelorism, and downslanting of palpebral fissures. B: Facial features of Sotos syndrome (SS) patient at 15 months of age with 11p15 KCNQ1OT1 demethylation (isolated). Note the frontal bossing, downslanting palpebral fissures, and anteverted nostrils. Major scoliosis at 9 years of age. Figure 3. Three patients with del(22)(qter) from group II (unclassified overgrowth syndromes). Note the minor facial features, including a large forehead and prominent chin. Both cases have, in addition, facial features and developmental delay, which are not usual findings of BWS. Conversely, two patients clinically diagnosed as SS had 11p15 abnormalities (one UPD and one KCNQ1OT1 demethylation) (Table IIIB, Fig. 2B). The two children had overgrowth, macrocephaly, advanced bone age, developmental delay, characteristic facial features, ventriculomegaly (1/2), severe scoliosis (1/ 2), and seizures (1/2). In addition, they did not have macroglossia, earlobe creases or pits, frontal nevus flammeus, abdominal wall defect, or hypoglycemia. These findings suggest considering NSD1 and 11p15 testing in children with atypical features of BWS or SS. The relationship between NSD1 and the 11p15 region is unknown, but NSD1 encodes a nuclear protein containing an SET domain involved in histone methylation and may play a role in establishing imprinting of the 11p15 region [Baujat et al., 2004]. In group II, patients were considered as having unclassified overgrowth disorders when no NSD1, 11p15 region, or GPC3 abnormality was identified. However, within this group, the systematic screening led to the identification of two 22 qter deletions. The two patients with del(22)(qter) have in common overgrowth and behavioral alterations. The wide phenotypic spectrum of del(22)(qter) syndrome has been reviewed elsewhere [Phelan et al., 2001]: mental retardation with severe verbal delay, hypotonia, large ears, normal-toaccelerated growth, and behavioral alterations. Minor facial features, suggestive of SS, have been also mentioned (pointed chin, dolichocephaly, seizures, recurrent ear infections, and highly þ þ (or hypertonia) ? þþ þþ (tube feeding: 40%) þ þ þþþ Deep fingernails Marked (carpal age higher than hand age) þ Camptodactyly Deep horizontal chin crease Large ears Wide face Micrognathia and later macrostomia Hoarse voice Flat occiput Hypertelorism þþþ Large Scoliosis Hands and feet Macroglossia Ear creases or posterior helical ear pits Facial nevus flammeus Neonatal period Feeding difficulties Hypoglycemia Jaundice Hypotonia þþþ (84%) Hypertelorism Downslanting palpebral fissures High cleft palate Prominent jaw Facial flushing Pointed chin þþþ Large head þþþ Dolichocephalic large head Prominent forehead Broad forehead þþþ þ þþþ Weaver syndrome þþþ þ þþþ (83%) Sotos syndrome Skeletal Advanced bone age Prenatal features Fetal overgrowth Hydramnios Postnatal overgrowth (growth>90th centile) Birth head circumference Craniofacial configuration Syndrome þþþ Coarse facies þþþ þ þþþ Simpson–Golabi– Behmel syndrome þþ þþþ (30%–50%) þ þþ þþþ (62%) þþþ (95%) þþþ (76%) þþ (66%) Prominent ears Ptosis Epicanthic folds Pointed chin Dolichocephaly þþ (95% accelerated growth) del(22)(qter ) syndrome 50% neonatal death þ þ þ þþ (Continued) þ (vertebral defects) Short, broad with variable Large, fleshy hand, fifth finger deformities Clinodactyly (14%) Complete transverse palmar Hypoplasia of nails crease Fingernail hypoplasia Mild ectodermal dysplasia þ (þ) þþ Prominent eyes with relative Hypertelorism infraorbital hypoplasia Prognathism Downslanting palpebral fissures Capillary nevus in the central Epicanthic folds forehead and eyelids Short nose Macrostomia Dental malocclusion Central groove of the lower lip Relatively small Protruding occiput þþþ þþþ þþþ (88%–91%) Beckwith– Wiedeman syndrome TABLE IV. Major Clinical and Molecular Findings in the Main Overgrowth Disorders ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) 9 ? þ (?) þ (l case) NSDI in_of cases (?) AD þ (8%) þ ? ? NSDI in 2/3 of cases (?) AD þ (cystic dysplasia) þ þ þ þ Suppernumerary (debated) þ (macroglossia and malocclusion) Simpson–Golabi– Behmel syndrome cryptorchidism hypospadias þ (6,5%) þþ (35%) þ þ þ þ þ þ þ (8%–8%, malignant, below 5 þ (malignant, below 5 years years) 11p15 anomalies in 80%–85% GPC3 (Xq25-q27) X-linked, Sporadic þþþ familial in 5% Mild expression in heterozygous females þ þþ (60%, cysts, hydronephrosis) Large ventricles þ þ þ Hydrocephaly þ þþ (50%) þþ 80% þþþ þ þ þþ (12.5%) þþ (60%) (debated) þ (macroglossia) Beckwith– Wiedeman syndrome þ þ þ þþ Weaver syndrome Possible þ þ þ þþ þ þþ Sotos syndrome The frequency of clinical findings approximatively estimated as: nearly constant (þþþ), frequent (þþ), occasionally reported (þ), absent (). Gene Inheritance Behavioral problems Neurological findings Ventriculomegaly CNS malformation Seizures Congenital renal abnormalities Genitalia Congenital heart defects Heart arrhythmias Polydactyly Cleft palate Neoplasms Abdominal wall defect Omphalocele Umbilical hernia Diastasis recti Hemihypertrophy Organomegaly Nipples Mental retardation Lack of fine motor skills Delay in speech Syndrome TABLE IV. (Continued) SHANK3 disruption þþþ þþþ(severe) þþ del(22)(qter ) syndrome 10 AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) ARTICLE ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) Figure 4. 11 Clinical overlap between Sotos, Weaver, Beckwith–Wiedemann, and Simpson–Golabi–Behmel syndromes. arched palate) (Table IV). The size of the deletion is variable from 130 kb to over 9 Mb; FISH analysis with the SHANK3 probe and/or genotyping by microsatellite DNA markers [Wilson et al., 2003] should probably be performed in unclassified overgrowth patients. 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