Genotype–phenotype correlation in 13q13.3–q21.3 deletion

European Journal of Medical Genetics 54 (2011) e489ee494

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Original article

Genotypeephenotype correlation in 13q13.3eq21.3 deletion Lucie Tosca a, b, c, *, Sophie Brisset a, b, François M. Petit b, d, Corinne Metay e, Stéphanie Latour a, Benoît Lautier a, Axel Lebas f, Luc Druart g, Olivier Picone h, Anne-Elisabeth Mas i, Sophie Prévot i, Marc Tardieu f, Michel Goossens e, Gérard Tachdjian a, b, c a

AP-HP, Histologie-Embryologie-Cytogénétique, Hôpital Antoine Béclère, Clamart F-92141, France Université Paris Sud, Le Kremlin-Bicêtre F-94275, France c INSERM U935, Villejuif F-94801, France d AP-HP, Biochimie, Hormonologie et Génétique, Hôpital Antoine Béclère, Clamart F-92141, France e AP-HP, Plateforme de Génomique IMRB 955, Hôpital Henri Mondor, Créteil F-94010, France f AP-HP, Neuropédiatrie, Hôpital Bicêtre, Le Kremlin-Bicêtre F-94275, France g Biomnis, Paris F-75014, France h AP-HP, Gynécologie Obstétrique, Hôpital Antoine Béclère, Clamart F-92141, France i AP-HP, Anatomie Pathologique, Hôpital Antoine Béclère, Clamart F-92141, France b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 December 2010 Accepted 10 June 2011 Available online 21 June 2011

Pure interstitial deletions of the long arm of chromosome 13 are correlated with variable phenotypes according to the size and the location of the deleted region. Deletions involving the 13q13q21 region are rare. In order to establish interstitial 13q genotypeephenotype correlation, we used high resolution 244K oligonucleotide array in addition to conventional karyotype and molecular (fluorescent in situ hybridization, microsatellite markers analysis) techniques in two independent probands carrying a deletion 13q13 to 13q21. First patient was a 3-year-old girl with mental retardation and dysmorphy carrying a 13q13.3q21.31 de novo deletion diagnosed post-natally. The second one was a fetus with de novo del(13)(q14q21.2) associated with first trimester increased nuchal translucency. We showed that specific dysmorphic features (macrocephaly, high forehead, hypertelorism, large nose, large and malformed ears and retrognathia) were correlated to the common 13q14q21 chromosomal segment. Physical examination revealed overgrowth with global measurement up to the 95th percentile in both probands. This is the second description of overgrowth in patients carrying a 13q deletion. Haploinsufficiency of common candidates genes such as CKAP2, SUGT1, LECT1, DCLK1 and SMAD9, involved in cell division and bone development, is a possible mechanism that could explain overgrowth in both patients. This study underlines also that cytogenetic analysis could be performed in patients with overgrowth. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: Chromosome 13 Interstitial deletion Genotypeephenotype correlation Oligonucleotide array-CGH

1. Introduction

Abbreviations: aCGH, array-comparative genomic hybridization; BRCA2, breast cancer type-2; CKAP2, cytoskeleton associated protein 2; CNV, copy number variations; DCLK1, doublecortin-like kinase 1; DWM, DandyeWalker malformation; FISH, fluorescence in situ hybridization; FOXO1A, alveolar rhabdomyosarcoma; GPC3, glypican 3; GTG, giemsa banding; LECT1, leukocyte cell derived chemotaxin 1; MRI, magnetic resonance imaging; NSD1, nuclear receptor binding SET domain protein 1; PCDH8-17, protocadherin 8-17; PCR, polymerase chain reaction; RB1, retinoblastoma 1; RHG, giemsa reverse banding; SMAD9, TGFb signalization pathway; SUGT1, suppressor of G2 allele of SKP1; WCP, whole-chromosome painting. * Corresponding author. Service d’Histologie-Embryologie-Cytogénétique, Hôpital Antoine Béclère, 157, rue de la Porte de Trivaux, 92141 Clamart Cedex, France. Tel.: þ33 (0) 1 45 37 49 23; fax: þ33 (0) 1 45 37 42 07. E-mail address: [email protected] (L. Tosca). 1769-7212/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmg.2011.06.004

In normal individuals, chromosome 13 is made up of about 114 Mb DNA sequences and represents 3.5e4% of the total genome [1]. Chromosome 13 contains 633 genes including 296 pseudogenes, 105 putative non-coding RNA and 9 miRNA [1]. Chromosome 13 has one of the lowest gene densities (6.5 genes per Mb) and contains a central region of 38 Mb where the gene density drops to only 3.1 genes per Mb. Nevertheless, several tumor suppressor genes are present such as breast cancer type-2 gene (BRCA2), alveolar rhabdomyosarcoma (FOXO1A) and retinoblastoma 1 (RB1). Proximal deletions of the long arm of chromosome 13 are rare. The specific “13q- syndrome” was established in 1969 [2]. The first patient was published in 1963 and described mental/growth retardation associated with retinoblastoma [3]. Phenotypes are

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variable according to the size and the position of deleted regions. Common clinical features in 13q deletion include moderate/severe mental and growth retardation, no specific dysmorphic traits, hand/foot/digital abnormalities and cerebral (neural tube defects, holoprosencephaly, corpus callosum agenesis), cardiac or renal malformations. To date, five studies included molecular characterizations of the 13q deletions by using array-Comparative Genomic Hybridization (aCGH) [4e8] but only one patient carried a proximal 13q deletion. Parental origin of deleted chromosomes was not currently investigated in these studies. In 2001, approximately 140 patients carrying a 13q deletion were described using different kinds of cytogenetic techniques [9]. However, 13q13q14 interstitial deletions are rarely reported. In the present study we described clinical and molecular data on two independent probands carrying a de novo 13q interstitial proximal deletion. Agilent 244K oligonucleotide array-CGH was used to characterize chromosome aberrations after conventional and molecular cytogenetic techniques (karyotype and fluorescent in situ hybridization). The parental origin in patient 1 was determined by analyzing polymorphic microsatellite markers. By comparing both patients we observed common clinical features related to the 13q14.2q21.2 common region that were not reported in the literature. 2. Material and methods 2.1. Patients Parent’s patient 1 family history was unremarkable. At the age of 22 months, the proband’s weight, height and head circumference were 15 kg (>95th percentile), 95 cm (>95th percentile) and 49.5 cm (>95th percentile), respectively. Parental heights were within the normal range with 170 cm and 177 cm, respectively. The girl showed psychomotor delay. The standing position was not acquired and limb weakness was noted. Her clinical examination was normal. At the age of 25 months, the child was not able to walk alone. Her way of walking was ataxic. Radiography of lumbar spine and pelvis was normal as well as fundoscopy (for retinoblastoma diagnosis) and brain magnetic resonance imaging (MRI). At the age of 33 months, she had delayed psychomotor development. Her weight, height and head circumference were 16 kg (95th percentile), 99.5 cm (>95th percentile) and 51 cm (>95th percentile), respectively. Brain MRI showed a thin corpus callosum. At the age of 3 years, her height was still >95th percentile. She presented

psychomotor/developmental delay and she also had behavioral disorders, mainly irritability. Walking was still abnormal due to limb weakness. However, she was able to walk without assistance. The following dysmorphic features were noted: a high forehead, large ears, hypertelorism, broad and prominent nasal bridge and thin upper lip (Fig. 1A). The second proband was the first pregnancy of a 24-year-old woman that was marked by increased nuchal translucency (3.4 mm for a cranio-caudal distance at 61 mm) during first-trimester ultrasound examination. Fetal biometry and morphology were considered normal. Amniotic fluid was sampled for chromosomal analysis at 18 weeks and showed a deletion on chromosome 13. After a normal ultrasound examination at 22 weeks and genetic counseling, parents decided to continue the pregnancy. The woman was referred to our prenatal center for the third-trimester ultrasound examination that revealed hydramnios (31 weeks þ 6 days). Fetal head circumference was 32 cm (>95th percentile) and biparietal diameter was 9.3 cm (>95th percentile). Maternal weight and height were 79 kg and 163 cm and paternal weight and height were 56 kg and 181 cm, respectively. These values are in the normal range. After genetic counseling and according to the French law, termination of pregnancy was performed at 34 weeks of gestation and a fetal autopsy was realized. The male fetus’ weight, height, head circumference and biparietal diameter were 2550 g (95th percentile), 47 cm (50th percentile), 32 cm (75th percentile) and 95 cm (>95th percentile), respectively. Physical examination showed cranio-facial dysmorphism with dolichocephalia, hypertelorism, interorbitary crease, large nose, large and malformed ears, retrognatism, short neck and pre-frontal edema (Fig. 1B). Physical examination showed a single palmar crease and clinodactyly on the left hand fifth finger. Internal examination and nervous system examination were normal. The placenta was eutrophic with normal aspect. 2.2. Conventional cytogenetic analysis Chromosome analyses were performed from uncultured and cultured amniotic cells, and from peripheral lymphocytes using standard procedures (RHG and GTG bandings). 2.3. Fluorescence in situ hybridization (FISH) FISH analyses were performed on amniotic cells and metaphase spreads from propositus lymphocytes. The whole-chromosome painting (WCP) probe specific for chromosome 13 was used

Fig. 1. A. Patient 1 at 3 years showing dysmorphisms: high forehead, large ears, hypertelorism, broad and prominent nasal bridge and thin upper lip. B. Fetus 2 at 34 weeks of gestation. Note dolichocephalia, hypertelorism, interorbitary crease, large nose, large and malformed ears, retrognatism, short neck and pre-frontal edema.

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according to manufacturer’s recommendations (Q-Biogene, Illkirch, France) as well as the subtelomeric 13q probe and the AneuVysion probe (LSI13 on 13q14 and LSI21 on 21q22.13q22.2) (Vysis, Downers Grove, IL). BACs clones specific for the 13q chromosomal regions were used: RP11-442J17 located at 13q12.11 (18.354.949e18.448.773, hg18), RP11-187L3 located at 13q12.11 (19.834.836e19.920.914, hg18), RP11-165D7 located at 13q14.2 (47.960.647e48.075.065, hg18) and RP11-424E21 located at 13q21.32 (65.400.827e65.415.270, hg18). BAC DNAs were indirectly labeled by nick-translation using a FITC-dUTP nucleotide or Rhodamine-dUTP nucleotide (Roche Diagnostics, Rungis, France).

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3. Results 3.1. Conventional cytogenetic analysis In patient 1, chromosomal analysis on lymphocytes showed an abnormal female 46,XX,del(13)(q13q21) karyotype with a deleted region on 13q (Supl Fig. 1A). In fetus 2, cytogenetic analyses on amniotic cells revealed an abnormal chromosome 13 with a deleted region on the long arm [46,XY,del(13)(q14q21)] (Supl Fig. 2B). Analyses of parents’ peripheral blood lymphocytes showed normal standard karyotypes in both families. 3.2. FISH analysis (data not shown)

2.4. Array-comparative genomic hybridization (aCGH) Genomic DNA was isolated from peripheral blood (patient 1) and from lung (fetus 2), using Qiagen DNeasy Blood and Tissue Kit (Qiagen, Courtaboeuf, France). The extracted DNA concentrations were estimated using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Extracted DNA was used for array-comparative genomic hybridization or for microsatellite analysis. The genomic imbalances of lung and lymphocyte cells were analyzed by aCGH using 244K oligonucleotide arrays (Hu-244A, Agilent Technologies, Massy, France). All array hybridizations were performed according to the manufacturer’s recommended protocols. Briefly, 2 mg of genomic DNA were digested with AluI (5 units) and RsaI (5 units) for 2 h at 37  C and fluorescently labeled with the Agilent Genomic DNA labeling kit PLUS (Agilent Technologies, Massy, France). Male or female human genomic DNA (Promega, Charbonnière, France) was used as reference. Experiments were done in dye-swap. Cy5-dUTP patient DNA and gender-matched reference DNA labeled with Cy3-dUTP were denatured and preannealed with Cot-1 DNA and Agilent blocking reagent prior to hybridization for 40 h at 20 rpm in a 65  C rotating hybridization oven (Agilent Technologies). After washing, the slides were scanned on an Agilent Microarray Scanner. Captured images were processed with Feature Extraction 9.1 software and data analysis was performed with CGH Analytics 3.5 (Agilent Technologies). The ADM-2 Genomic Work Bench algorithm was used for statistical analysis. Copy number variations (CNV) were considered significant if they were defined by 3 or more oligonucleotides spanned at least 8.9 kb, contained at least one gene and were not identified in The Database of Genomic Variants. 2.5. Microsatellite analysis Seven polymorphic markers from chromosome 13 were coamplified by multiplex polymerase chain reaction (PCR): D13S171 (13q13.1), D13S219 (13q13.3), D13S218 (13q13.3), D13S263 (13q14.11), D13S153 (13q14.2), D13S163 (13q21.1) and D13S1320 (13q21.31). Primer sequences were designed according to GeneDB locus description from The Wellcome Trust Sanger Institute. Multiplex PCR was carried out using the ABI Prism True Allele PCR Premix kit, using 50 ng of DNA in a 15 ml reaction volume and fluorescence dye labeled primers (5 mM each primer). PCR conditions were initial denaturation at 95  C for 12 min followed by 10 cycles of 94  C for 15 s, 55  C for 15 s, 72  C for 30 s, 20 cycles of 89  C for 15 s, 55  C for 15 s, 72  C for 30 s, and final 72  C extension step for 10 min (Veriti Thermal cycler, Applied Biosystems, Courtaboeuf, France). PCR products were then separated onto an ABI Prism 3130 analyzer (AppliedBiosystems) with the GeneScan 500LIZ as size standard. Data were analyzed using the GeneMapper 4.0 software (AppliedBiosystems, Courtaboeuf, France).

In patient 1, WCP13 probe showed complete hybridization on normal chromosome 13 and on the abnormal chromosome 13. No hybridization signal was detected on any other chromosome. This finding excluded the possibility of an insertion or a translocation. FISH studies also showed LSI13q14 deletion including RB1 locus. The subtelomeric 13q probe showed hybridization on the normal and the abnormal chromosome 13 underlying an interstitial deletion. BACs RP11-187L3 (13q12.11), RP11-442J17 (13q12.11), and RP11-424E21 (13q21.32) gave one signal on normal chromosome 13 and one signal on deleted chromosome 13. No hybridization signal of these BACs probes were detected on any other chromosome. BAC RP11-165D7 (13q14.2) showed one signal on normal chromosome 13 and no signal on the deleted one. In fetus 2, FISH experiments on uncultured and on cultured amniocytes showed a deletion in the 13q14 region (LSI13q14 RB1 probe). In both patients, FISH analysis on parents cultured lymphocytes using the LSI13q14 RB1 probe showed normal signals.

3.3. Array-comparative genomic hybridization The aCGH experiment showed in the first patient a 25.27 Mb loss of the 13q13.3q21.31 region (Fig. 2A). The analysis revealed a proximal breakpoint located on 13q13.3 (position 36.275.040) and a distal breakpoint on 13q21.31 (position 61.552.601) including 216 genes. For second proband, a 12.87 Mb loss of the 13q14.2q21.2 region was observed (Fig. 2B). Proximal breakpoint was located on 13q14.2 (position 46.974.739) and distal breakpoint on 13q21.31 (position 59.845.632) including 103 genes. These genomic positions were determined using the version 18 of the Human genome built (http://genome.ucsc.edu/). Analyses revealed other variations (gain or loss) on chromosome 13 and on the other chromosomes. These changes corresponded to CNV previously reported in the database of genomic variants (http:// projects.tcag.ca/variation/). 3.4. Microsatellite analysis In patient 1 segregation analysis showed that D13S171, D13S219 and D13S1320 markers were non informative whereas D13S218, D13S263, D13S153 and D13S163 microsatellite markers were consistent with paternal origin (Supl Table 1). This analysis was not performed for family 2, since parental DNA was not available. In summary, these conventional and molecular cytogenetic experiments allowed a precise characterization of the chromosomal aberration in both patients. Patient 1 carried a pure 25.27 Mb del(13)(q13.3q21.31)dn of paternal origin and patient 2 had a pure 12.87 Mb del(13)(q14.2q21.2)dn. Thus, the deleted region shared by both patients was from 13q14.2 to 13q21.2.

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Fig. 2. Chromosome 13 profile from array-based CGH analysis showing loss (deletion) for DNA sequences located in 13q13.3q21.31 region (25.27 Mb) for patient 1 (A) and in 13q14.2q21.2 region (12.87 Mb) for fetus 2 (B).

4. Discussion In the present study, interstitial deletions of the 13q13q21 region in two unrelated patients were characterized. The probands, carrying deletions of 25.27 and 12.87 Mb respectively, had several clinical features in common: specific dysmorphy, macrocephaly and overgrowth but also individual ones such as single transverse palmar crease or hand malformations. In 2008, about 150 patients carrying pure 13q deletion were described using different cytogenetic techniques. However, 13q proximal and constitutional deletions including 13q13 and 13q14 regions were rarely reported [4,10e13] (Table 1). We will focus this discussion on proximal 13q deletion overlapping the 13q13q21 region. Reported patients with proximal 13q deletion included a 11-year-old girl with mild mental retardation, dysmorphic features, increased occipitofrontal head circumference and skeletal anomalies carrying a 13q14.11q21.31 deletion [4]. Fukushima et al. reported two familial cases carrying a del(13)(q14.1q21.2) associated with retinoblastoma and dysmorphic traits [12] and Motegi study reported a patient with deletion extended from 13q14 to 13q21 or 13q22 associated with retinoblastoma and dysmorphy [10]. Finally, Skrypnyk and Bartsch in 2004 reported a 10-year-old boy with a del(13)(q14q14) showing mild overgrowth, hepatomegaly, inguinal hernia, cryptorchism, mild developmental delay and retinoblastoma [13]. The phenotype of patients carrying a proximal or distal 13q deletion is considered as unspecific by most authors [4]. In our two patients, broad forehead is associated with hypertelorism, large and malformed ears and retrognathia. To our knowledge, these dysmorphic features were not specifically reported together in previous

studies and thus could be correlated with the 13q14.2q21.2 chromosomal segment. Indeed, Fukushima et al. reported a high forehead, low and broad nasal root, a short and bulbous nose, a long philtrum, and open mouth with a thin upper lip, and prominent earlobes [12]. The study of Mogeti indicated prominent eyebrows, broad nasal bridge, bulbous tip of the nose, a large mouth with a thin upper lip, and a long philtrum [10]. Our study is the second work that underlined overgrowth in patients associated with interstitial chromosome 13q deletion. Skrypnyk and Bartsch reported a 10-year-old boy with a 370e450 Kb del(13)(q14q14) of maternal origin (explored by karyotype, FISH using LSIRB1 probe and microsatellite markers) [13]. The patient showed mild overgrowth, hepatomegaly and inguinal hernia. The boy had also cryptorchism, mild developmental delay, bifocal retinoblastoma and pinealoma. Thus, the present work and previous reported data [13] demonstrate that the deletion 13q14 may be associated with overgrowth when deleted. The well known overgrowth syndromes include SimpsoneGolabi syndrome encoded by glypican 3 (GPC3 gene located on Xq26 chromosomal region), BeckwitheWiedemann syndrome (genetic/ epigenetic modifications on 11p15), Weaver syndrome [nuclear receptor binding SET domain protein 1 (NSD1) mutation on 5q35], Sotos syndrome (NSD1 deletion, duplication or mutation on the same 5q35 segment) and Perlman syndrome (the involved region remains unknown). Our present observations allow a genotypeephenotype correlation on chromosome 13 proximal regions as summarized in Table 1. To our knowledge, the present study is the second report of proximal and interstitial 13q deletions including the 13q14 region

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Table 1 Phenotypes in patients published with a pure 13q proximal deletion including present study. References

[4] Case 2

Age Region Inheritance Deletion size Parental origin Sex Mental retardation Behavioral disorders Growth Head circumference High nasal bridge Long philtrum Micrognathia Nose Thin upper lip Macroglossia High forehead Hypertelorism Height/narrow palate Ear malformation Retinoblastoma Skeletal anomalies Brain anomalies Hepatomegaly Inguinal hernia Cryptorchism Optic nerve hypoplasia Hand or feet anomalies Others

11 years 13q14.11q21.31 nr 22.8 Mb nr F þ nr nr þ þ nr e nr nr e nr e þ þ nr þ e nr nr e þ e e

Molecular techniques

[10]

9 months 13q12q22 De novo nr nr F þ e e nr þ nr þ Small/bulbous þ nr nr nr nr nr þ þ þ nr nr e nr nr Tetralogy of Fallot and hirsutism Spectral genomic e 1 Mb chip

[12] Mother

[12] Son

[13]

Present study Patient 1

Fetus 2

33 years 13q14.1q21.2 De novo nr nr F e e nl nl þ þ nr Bulbous þ e þ nr nr þ þ þ nr nr nr nr nr þ e

21 months 13q14.1q21.2 Maternal nr Maternal M þ e nl nl þ þ nr Small/bulbous þ e þ nr nr þ þ þ nr nr nr nr nr þ e

3 years 13q13.3q21.31 De novo 25.27 Mb Paternal F þ þ þ þ þ e þ nr þ e þ þ e þ e þ þ e e e nr e e

34 weeks of gestation 13q14.2q21.2 De novo 12.87 Mb na M na na þ þ e e þ Large e e þ þ e þ nr e e e e e nr þ e

e

e

10 years 13q14q14 De novo 400 kbe4 Mb Maternal M þ e þ þ þ nr nr nr e þ nr nr nr þ þ nr þ þ þ þ nr þ Bulging abdomen and liver enlargement Microsatellite markers

Microsatellite markers Microsatellite markers and 244K array-CGH and 244K array-CGH

na, not available; nl, normal; nr, not reported.

that used a high resolution technique. The first report was case 2 of the Ballarati study with a 13q14.11q21.31 deletion [4]. Parental origin of 13q14 deletions has not been studied thus far and could only be investigated in our patient 1. Thus no information on a parent-of-origin effect is available at the moment. In our study, the first patient 13q deletion encompassed 216 genes and the second one 103 genes. Common ones included critical genes for cell growth and bone development that could explain patients overgrowth such as cytoskeleton associated protein 2 (CKAP2, mitotic progression), suppressor of G2 allele of SKP1 (SUGT1, G1/S and G2/ M cell cycle transitions), RB1 and leukocyte cell derived chemotaxin 1 (LECT1, bone development). Indeed, a modification of cell cycle regulation (on mitosis stage or on critical check-points) by altering CKAP2 and SUGT1 gene expression could induce an elevation of mitotic index and thus induce tissues overgrowth. Haploinsufficiency of LECT1 gene (also named Chondromodulin-I) that is involved in endochondral bone development may be responsible of overgrowth too [14]. Moreover, deletion of protocadherin 8-17 (PCDH8-17) that regulates brain cell connections could be a candidate to explain patient 1 mental retardation. These genes encode integral membrane proteins that function in cell adhesion in a central nervous system-specific manner [15]. Thus, we can speculate that misregulation of PCDH8-17 expression gene could affect neurone communication thus inducing mental retardation. Other genes involved in cell growth are specific to patient 1 larger deletion such as doublecortin-like kinase 1 (DCLK1, cell division with a coding protein associated to microtubules), TGFb signalization pathway, cell proliferation (SMAD9), and C13orf15 locus under p53 regulation (cell proliferation). Indeed, DCLK1 gene encodes a protein which binds microtubules, regulates microtubule polymerization and is involved in several different cellular processes

including neuronal migration, retrograde transport, neuronal apoptosis and neurogenesis [16]. Furthermore, alteration of SMAD9 gene and C13orf15 locus expression could be responsible of cell cycle misregulation inducing aberrant activation of cell growth. Thus, we can speculate that haploinsufficiency of one or more of these genes could be responsible for overgrowth in individuals carrying 13q14q21 deletion. Further studies are needed to define whether a one-gene haploinsufficiency mechanism could be associated with our described phenotype. Finally, in both present patients, the deletion on chromosome 13 involved the 13q14 region including RB1 gene. Retinoblastoma is due to haploinsufficiency of the retinoma susceptibility RB1 gene located at 13q14.2 [17]. Individuals with mutations [18,19] or deletions [20] of the RB1 gene are at high risk for retinoblastoma which is often bilateral and/or multifocal. These patients are also at risk for osteosarcomas, pinealoblastomas and other sarcomas [10,12,13,20]. Prognosis is dependent on the timing of diagnosis. After genetic counseling, first patient underwent regular ophtalmologic evaluations that, until now, were normal. This is in accordance with the fact that a relevant proportion of patients with 13q14 band deletion does not develop tumor at all [17,21]. 5. Conclusions In conclusion, this is the second report of patients with 13q proximal deletion including 13q14 locus having remarquable overgrowth and macrocephaly but the first one using high resolution cytogenetic technique. Moreover, specific dysmorphic traits such as high forehead, hypertelorism, large and malformed ears and retrognathia were correlated also with the 13q14.2q21.2 chromosomal segment.

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The presented patients were submitted on the web-based database ECARUCA (http://agserver01.azn.nl:8080/ecaruca/ecaruca.jsp). Patient 1 was registered as ID4839 number and Fetus 2 as ID4850 number. Acknowledgments We thank L. Lecerf, A. Briand and the technical team for CGH array experiments help and results interpretation. We also thank the DHOS (Direction de l’Hospitalisation et de l’Organisation des Soins) for their support in the development array-CGH platform Paris Sud.

[8]

[9]

[10]

[11]

[12]

Appendix. Supplementary material Supplementary material related to this article can be found online at doi:10.1016/j.ejmg.2011.06.004.

[13]

[14]

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