European Journal of Medical Genetics 49 (2006) Original article

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1 European Journal of Medical Genetics 49 (2006) Original article Prader-Willi-like phenotype: investigation of 1p36 deletion in 41 patients with delayed psychomotor development, hypotonia, obesity and/or hyperphagia, learning disabilities and behavioral problems Carla S. D Angelo a, *, José A. Da Paz b, Chong A. Kim c, Débora R. Bertola c, Claudia I.E. Castro a, Monica C. Varela a, Célia P. Koiffmann a a Human Genome Study Center, Department of Genetics and Evolutive Biology, Institute of Biosciences, University of São Paulo, São Paulo, SP, Brazil b Child Neurology Service, Department of Neurology, Hospital das Clínicas, University of São Paulo School of Medicine, São Paulo, SP, Brazil c Clinical Genetics Unit, Child s Institute, Hospital das Clínicas, University of São Paulo School of Medicine, São Paulo, SP, Brazil Available online 10 March 2006 Abstract Monosomy 1p36 is one of the most commonly observed mental retardation (MR) syndromes that results in a clinically recognizable phenotype including delayed psychomotor development and/or MR, hypotonia, epilepsy, hearing loss, growth delay, microcephaly, deep-set eyes, flat nasal bridge and pointed chin. Besides, a Prader-Willi syndrome (PWS)-like phenotype has been described in patients with 1p36 monosomy. Forty-one patients presenting hypotonia, developmental delay, obesity and/or hyperphagia and behavioral problems who tested negative for PWS were investigated by FISH and/or microsatellite markers. Twenty-six were analyzed with a 1p-specific subtelomeric probe, and one terminal deletion was identified. Thirty patients (15 of which also studied by FISH) were investigated by microsatellite markers, and no interstitial 1p36 deletion was found. Our patient presenting the 1p36 deletion did not have the striking features of this monosomy, but her clinical and behavioral features were quite similar to those observed in patients with PWS, except for the presence of normal sucking at birth. The extent * Corresponding author. Tel.: ; fax: address: cdangelo@ib.usp.br (C.S. D Angelo) /$ - see front matter 2006 Elsevier Masson SAS. All rights reserved. doi: /j.ejmg

2 452 C.S. D Angelo et al. / European Journal of Medical Genetics 49 (2006) of the deletion could be limited to the most terminal 2.5 Mb of 1p36, within the chromosomal region 1p p36.32, that is smaller than usually seen in monosomy 1p36 patients. Therefore, chromosome 1p36.33 deletion should be investigated in patients with hypotonia, developmental delay, obesity and/or hyperphagia and behavioral problems who test negative for PWS Elsevier Masson SAS. All rights reserved. Keywords: 1p36 Monosomy; Prader-Willi syndrome; Subtelomeric rearrangement; Syndromic obesity; MLPA 1. Introduction Cryptic subtelomeric chromosomal rearrangements occur in ~5% of patients with idiopathic mental retardation (MR) [6]. Terminal deletions of 1p are the most commonly observed monosomy in screenings for detecting submicroscopic subtelomeric abnormalities [7]. Deletion of the most telomeric band of the short arm of chromosome 1 (1p36) results in a clinically recognizable syndrome, named monosomy 1p36 syndrome, affecting 1 out of 5000 newborns [19]. The main characteristics include delayed psychomotor development and/or MR, hypotonia, hearing loss, visual anomalies, failure to thrive, epilepsy, microcephaly, brachycephaly, deep-set eyes, flat nasal bridge and pointed chin. Feeding difficulties in infancy are also common in these patients and some of them develop hyperphagia and/or obesity, suggesting a phenotypic overlap with Prader-Willi syndrome (PWS) [8 10,13,20,21,23]. A PWS-like phenotype was also described in patients with 6q and 9q chromosome deletions [2,5]. The monosomy 1p36 syndrome occurs as a consequence of a diversity of chromosome abnormalities frequently undetectable by routine chromosome studies, including pure terminal deletions, terminal deletions associated with cryptic interrupted inverted duplications, derivative chromosomes, interstitial deletions and complex rearrangements [1,7,8]. The remarkable variability in the clinical presentation of the syndrome results from the different extent of the deletions, ranging from 1.5 to over 10.5 Mb, and chromosome rearrangements. The breakpoints were described at different locations of the chromosome segment 1p36, not showing preferential clustering within a common interval. Although genes were not yet identified, critical regions are found in the most distal segment of 4.0 Mb [8]. Few patients (11.3%) were reported with terminal deletion of less than 2.5 Mb, however, genotype phenotype correlations were not established so far [8]. Herein we report a patient with a subtelomeric deletion of 1p restricted to the most terminal 2.5 Mb of 1p36.33, and a Prader-Willi-like phenotype, diagnosed amongst 41 patients presenting hypotonia, developmental delay, obesity and/or hyperphagia, learning disabilities and behavioral problems, who tested negative for PWS. 2. Materials and methods 2.1. Subjects Forty-one patients presenting delayed psychomotor development, hypotonia, obesity and/or hyperphagia, learning disabilities and behavioral problems were evaluated. These patients were referred to our laboratory for genetic testing for PWS by physicians of the Neurology and Clinical Genetics Unit of the Child s Institute of Hospital das Clínicas of the University of

3 São Paulo School of Medicine, and were examined by at least one of the authors, following a standard protocol that included evaluation of physical and behavioral characteristics. Informed consent was obtained for all patients from parents or legal guardians Methods Cytogenetic and FISH studies Chromosome studies of the patients were performed in cultivated peripheral blood lymphocytes, using the GTG-banding technique to investigate structural and numerical alterations. Subtelomeric FISH for region 1p was performed in metaphase chromosomes of 26 patients whose cultivated lymphocytes were available using the TelVysion 1p probe (Vysis) which contains a locus (CEB108/T7) estimated to be within 300 kb of the end of the chromosome or TEL 1p (Oncor) also within 300 kb of the 1p telomere Molecular studies Genomic DNA was extracted from peripheral blood leukocytes by a standard salting out method. PWS diagnosis was ruled out by methylation pattern analysis of the SNURF-SNRPN exon 1 amplified by PCR [24]. Investigation of the 1p deletion was performed by microsatellite analysis of three different polymorphic markers: D1S243 mapped at 2.2 Mb per pter (1p36.33); D1S171 at 2.5 Mb from the 1p telomere (1p36.32) and D1S468 at 3.6 Mb per pter (1p36.32), approximately (based on the UCSC Genome Browser). Primer sequences and PCR conditions were obtained at The Genome Database ( Multiplex ligation-dependent probe amplification (MLPA) A specifically designed set of probes for testing for subtelomeric chromosomal imbalances, the SALSA P036 Human Telomere MLPA kit (MRC-Holland, Amsterdam, Netherlands), was used for subtelomere screening on the patient detected with the 1p36 deletion. To ascertain the extent of the deletion in the 1p36 region, MLPA was performed with the SALSA MLPA kit P147 1p36 deletions which contains 36 probes in 1p36 and nine control probes on others chromosomes (MRC-Holland, Amsterdam, Netherlands). DNA denaturation and hybridization, ligation and PCR reactions were carried out according to the manufacturer s instructions, as described in Schouten et al. [18]. 3. Results C.S. D Angelo et al. / European Journal of Medical Genetics 49 (2006) All cases presented normal karyotypes. Twenty-six of the 41 patients were analyzed with the 1p-specific subtelomeric probe (Fig. 1) that showed normal hybridization patterns (patients 1 25), except in one patient (patient 41) who presented a de novo 1p36 terminal deletion {46, XX.ish del(1)(p36)([telvysion1p]-), Fig. 2}, since her parents showed normal karyotypes and FISH analyses. MLPA analysis with the SALSA P036 Human Telomere kit showed a terminal deletion of 1p, with no other detectable subtelomeric rearrangement (Fig. 3). The breakpoint of the deletion was located by MLPA kit P147 between probes 4678-L4057 and 1122-L0680 that detects the genes PEX10 (~2.4 Mb 1pter) and FLJ10782 (~2.48 Mb 1pter), respectively (Fig. 4). So, the extent of the deletion could be limited to the most terminal 2.5 Mb of 1p36, within the chromosomal region 1p p The identification of the parental origin of

4 454 C.S. D Angelo et al. / European Journal of Medical Genetics 49 (2006) Fig. 1. Analysis of microsatellite markers and/or subtelomeric FISH probe (41 patients). Polymorphic markers and FISH probe are listed from distal (top) to proximal chromosome 1p36 region. Fig. 2. FISH study of the patient with the specific 1p subtelomeric probe (TelVysion 1p, Vysis). Note missing signal on one homolog 1 (arrow) and normal signal on the other (arrow). the deletion was not possible, since the study with the most distal marker D1S243 was uninformative and biparental inheritance was disclosed with markers D1S171 and D1S468. Thirty of the remaining 40 patients (15 of which were also studied by FISH) were investigated for deletions using microsatellite markers: 23 were heterozygous for the D1S243 marker and 25 for the D1S468 marker. Eleven cases were uninformative for one or both markers;

5 C.S. D Angelo et al. / European Journal of Medical Genetics 49 (2006) Fig. 3. Detection of 1p subtelomeric deletion by MLPA. Control DNA (A) and DNA from our patient with the 1p36 deletion (B). The arrow indicates the position of the aberrant MLPA probe (CAB45). Fig. 4. The extent of the deleted chromosome segment investigated by MLPA kit P147. Control DNA (A) and DNA from our patient with the 1p36 deletion (B). The arrows indicate the deleted probes ( C means control probes).

6 456 C.S. D Angelo et al. / European Journal of Medical Genetics 49 (2006) these patients were also studied with the D1S171 marker that revealed biparental inheritance in eight cases and was uninformative in three cases (Fig. 1). Therefore, molecular investigation with microsatellite markers excluded terminal deletions less than 2.2 Mb pter in 11 patients, found to be heterozygous for marker D1S243 (patients 26 36); in the remaining cases which were also heterozygous for marker D1S243 (patients 11 22), a terminal deletion was ruled out by FISH. Four patients were not investigated by FISH and presented uninformative results for the most distal marker D1S243 (patients 37 40). Three of them were also uninformative for at least one of the other markers studied (patients 38 40). No interstitial 1p36 deletions were identified amongst the 30 studied patients Case report The patient with the de novo 1p36 terminal deletion, a 13-year-old girl, was the first child of young, non-consanguineous, healthy Brazilian parents. The family history was unremarkable. She was born at term after an uneventful pregnancy. Birth weight was 2350 g (< 3rd centile) and length 45 cm (< 3rd centile). Congenital bilateral cataract was diagnosed at birth. She presented neonatal hypotonia with normal sucking. Development was delayed: she could walk at 2 years and 2 months and acquired language at 4 years and 6 months. Hyperphagia and obesity began at 3 years of age, associated with crying episodes and excessive irritability. She was hyperactive from the age of 3 to the age of 6 years. Physical examination at 12 years of age revealed a weight of 56 kg (> 97th centile), a height of 152 cm (75 < p < 90th centile), and a head circumference of 53 cm (50th centile). She had a peculiar facies, with a high and prominent forehead, narrow bitemporal diameter, thick eyebrows with mild synophrys, slightly deep-set eyes, downslanting palpebral fissures, posteriorly rotated ears, prominent cheeks and a small mouth (Fig. 5). Hypertrichosis and bilateral fifth-finger clinodactyly were also noted. Cardiac auscultation, hearing evaluation, cra- Fig. 5. The patient at 12 years of age. Note high and prominent forehead, narrow bitemporal diameter, downward slanting palpebral fissures, slightly deep-set eyes.

7 nial MRI and EEG were normal. On ophthalmologic evaluation, she presented myopia and astigmatism; a thyroid US revealed the presence of nodules. Her plasmatic levels of total cholesterol were elevated, but the TSH levels were normal. She never had seizures. At the age of 13 years she developed diabetes. The patient presented only syllabic reading, but acquired complex language and presented characteristics of the attention deficit disorder. Skin picking was reported. 4. Discussion C.S. D Angelo et al. / European Journal of Medical Genetics 49 (2006) In an ongoing research on syndromic obesity (obesity, phenotypic abnormalities and MR [3]), 41 patients presenting hypotonia, developmental delay, obesity and/or hyperphagia, learning disabilities and behavioral problems were studied by FISH and/or microsatellite markers to investigate 1p36 deletions. These studies were done after exclusion of PWS by the specific genetic test, performed in all patients (data not shown). The FISH study identified a subtelomeric terminal deletion of 1p in one out of 26 patients. Furthermore, microsatellite marker analyses ruled out the presence of terminal deletions of less than 2.2 Mb in 11 patients and of interstitial deletions in 30 patients. In four patients the molecular results were inconclusive. The main clinical findings of the studied patients are presented in Table 1. The patient with the de novo 1p36 terminal deletion, a 13-year-old girl, presented delayed psychomotor development, neonatal hypotonia, obesity, hyperphagia, behavioral problems, learning disabilities, skin picking, narrow bitemporal diameter, which are all features com- Table 1 Clinical findings in the reported patients with PWS-like phenotype and in the patient identified with a terminal 1p deletion Clinical feature Reported patients (N = 40) Present case Number of patients with the feature/ total evaluated (%) Sex ratio (male:female) 25:15 F Age (average) (years) 7 years 10 months 13 years Range 12 month 15 years Birth weight (average) (g) Range g Developmental delay 40/40 (100) + Obesity and/or hyperphagia 40/40 (100) + Learning disabilities 28/28 (100) a + Behavioral problems 28/32 (87.5) + Hypotonia 22/32 (68.75) + Seizures 19/37 (51.35) Feeding problems 14/35 (40) Height (centile) < 3 8/39 (20.51) /39 (30.76) /39 (30.76) > 97 7/39 (17.94) OFC (centile) < 2 4/38 (10.52) /38 (44.73) /38 (13.15) > 98 12/38 (31.57) a Patients older than 6 years of age.

8 458 C.S. D Angelo et al. / European Journal of Medical Genetics 49 (2006) monly observed also in the PWS; she also had slightly deep-set eyes and visual anomalies, characteristics described in patients with the monosomy 1p36 syndrome, although the striking facial appearance of this syndrome was absent. Our patient had also congenital bilateral cataract, diabetes and thyroid nodules, which are uncommon findings in 1p36 deletion cases (Table 2). Derivative chromosomes in which the 1p telomeric region was replaced by another chromosomal end were described in 17% of the patients, most of them cryptic and not inheritable [8]. Therefore, MLPA was used for subtelomere screening and no subtelomeric rearrangement other than the 1p deletion was detected. The extent of the deleted chromosome segment in our patient was limited to the most terminal 2.5 Mb of 1p36.3 as was demonstrated by MLPA with a specific designed set probes for testing for 1p36 deletions (SALSA MLPA kit P147). Therefore, our patient s deletion (< 2.5 Mb) is smaller than those usually seen in monosomy 1p36 patients. Heilstedt et al. [8] reported that amongst 53 terminal deletions only six (11.3%) had less than 2.5 Mb of extension. Although critical regions had been localized within the most distal 4.0 Mb segment of 1p36.3, full genotype phenotype correlations were not established [8]. Furthermore, Torisu et al. [22] reported one of the smallest 1p deletions, with the breakpoint at Mb pter, in a patient presenting the striking features and facial appearance of the monosomy 1p36 syndrome. As our patient presents an atypical facies, we suggest that the critical region for the facial appearance of the monosomy 1p36 syndrome is located distal to marker D1S468 and proximal to the PEX10 gene, at Mb of the 1p telomere. Obesity and/or hyperphagia are features already described in patients with 1p36 deletion [4, 9,15 17,20,23], but correlations between the extents of the deleted chromosomal segments were not provided. Neumann et al. [13] described a patient with a mild but typical monosomy Table 2 Clinical characteristics of monosomy 1p36 syndrome reported in the literature and in our patient Clinical feature Number of patients with the feature/ Our patient total evaluated (%) Developmental delay/mr 30/30 (100) [8] + Hypotonia 26/30 (87) [8] + Growth delay (postnatal) 11/13 (85) [20] Feeding difficulties in infancy 19/30 (63) [8] Obesity 2/13 (15) [20] + Epilepsy 15/31 (48) [8] Hearing loss 23/28 (82) [8] Hypermetropia 20/30 (67) [8] Hypothyroidism 6/30 (20) [8] Orofacial clefting anomalies 5/30 (17) [8] Structural heart defects 13/30 (43) [8] Dilated cardiomyopathy 7/30 (23) [8] Microcephaly 18/30 (60) [8] Brachycephaly 18/30 (60) [8] Deep-set-eyes 24/30 (80) [8] + Flat nasal bridge 23/30 (77) [8] Flat nose 20/30 (67) [8] Pointed chin 20/30 (67) [8] Thickened ear helices 16/30 (53) [8] Asymmetric ears 16/30 (53) [8] Short fifth finger 26/30 (87) [8] Heilstedt et al. [8], Shapira et al. [20].

9 1p36 phenotype that presented obesity and hyperphagia with a terminal deletion of 1p and a breakpoint between 1.6 and 3.1 Mb pter. Our patient s breakpoint was narrowed down to between 2.4 and 2.48 Mb pter. Therefore, we suggest that the chromosome segment 1p is a critical region for the manifestation of obesity and hyperphagia. To our knowledge, congenital cataract was not previously described in patients with monosomy 1p, but cataracts were reported in a 47-year-old patient [14] and in two children [11]. One form of congenital cataract (OMIM ) was mapped to the chromosome segment 1pter p36.13 [12], and autosomal dominant inheritance with variable expressivity was suggested. Concluding, we suggest that monosomy 1p36 syndrome should be considered in patients with hypotonia, developmental delay, obesity, hyperphagia, behavioral disorders, learning difficulties and a negative genetic test for PWS, even in the absence of the striking facial features. Acknowledgements This work was supported by FAPESP (C.S.D. 01/ ), CEPID-FAPESP, CAPES and CNPq. We thank Roseli M. Zanelato for technical assistance and Dr. Andrew Wallace for the spreadsheets used in MLPA data analyses. References C.S. D Angelo et al. / European Journal of Medical Genetics 49 (2006) [1] B.C. Ballif, W. Yu, C.A. Shaw, C.D. Kashork, L.G. Shaffer, Monosomy 1p36 breakpoint junctions suggest premeiotic breakage fusion bridge cycles are involved in generating terminal deletions, Hum. Mol. Genet. 12 (17) (2003) [2] V. Cormier-Daire, F. Molinari, M. Rio, O. Raoul, M.-C. de Blois, S. Romana, M. Vekemans, A. Munnich, L. Colleaux, Cryptic terminal deletion of chromosome 9q34: a novel cause of syndromic obesity in childhood?, J. Med. Genet. 40 (2003) [3] M.-A. Delrue, J.L. Michaud, Fat chance: genetic syndromes with obesity, Clin. Genet. 66 (2004) [4] E.A. Eugster, S.A. Berry, B. Hirsch, Mosaicism for deletion 1p36.33 in a patient with obesity and hyperphagia, Am. J. Med. Genet. 70 (1997) [5] L. Faivre, V. Cormier-Daire, J.M. Lapierre, L. Colleaux, S. Jacquemont, D. Genevieve, P. Saunier, A. Munnich, C. Turleau, S. Romana, M. Prieur, M.C. de Blois, M. Vekemans, Deletion of the SIM1 gene (6q16.2) in a patient with a Prader-Willi-like phenotype, J. Med. Genet. 39 (2002) [6] J. Flint, S. Knight, The use of telomere probes to investigate submicroscopic rearrangements associated with mental retardation, Curr. Opin. Genet. Dev. 13 (2003) [7] H.A. Heilstedt, B.C. Ballif, L.A. Howard, C.D. Kashork, L.G. Shaffer, Population data suggest that deletions of 1p36 are a relatively common chromosome abnormality, Clin. Genet. 64 (2003) [8] H.A. Heilstedt, B.C. Ballif, L.A. Howard, R.A. Lewis, S. Stal, C.D. Kashork, C.A. Bacino, S.K. Shapira, L.G. Shaffer, Physical map of 1p36, placement of breakpoints in monosomy 1p36, and clinical characterization of the syndrome, Am. J. Hum. Genet. 72 (5) (2003) [9] K.M. Keppler-Noreuil, A.J. Carrol, W.H. Finley, S.L. Rutledge, Chromosome 1p terminal deletion: report of new findings and confirmation of two characteristic phenotypes, J. Med. Genet. 32 (1995) [10] E. Knight-Jones, S. Knight, H. Heussler, R. Regan, J. Flint, K. Martin, Neurodevelopmental profile of a new dysmorphic syndrome associated with submicroscopic partial deletion of 1p36.3, Dev. Med. Child Neurol. 42 (3) (2000) [11] S.J.L. Knight, R. Regan, A. Nicod, S.W. Horsley, L. Kearney, H. Tessa, R.M. Winter, P. Bolton, J. Flint, Subtle chromosomal rearrangements in children with unexplained mental retardation, Lancet 354 (1999) [12] A.M. Lund, H. Eiberg, T. Rosenberg, M. Warburg, Autosomal dominant cataract; linkage relations; clinical and genetic heterogeneity, Clin. Genet. 41 (1992) [13] B.L.M. Neumann, T. Polster, T. Spantzel, O. Bartsch, Unexpected death of a 12-year-old boy with monosomy 1p36, Genet. Counsel. 15 (1) (2004) [14] O. Reish, S.A. Berry, B. Hirsch, Partial monosomy of chromosome 1p36.3: characterization of the critical region and delineation of a syndrome, Am. J. Med. Genet. 59 (1995)

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