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Clinical and molecular delineation of a 16p13.2p13.13 microduplication
European Journal of Medical Genetics 58 (2015) 194e198
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Array report
Clinical and molecular delineation of a 16p13.2p13.13 microduplication E. Tassano a, *, M.G. Alpigiani b, A. Calcagno b, P. Salvati b, L. De Miglio b, P. Fiorio a, C. Cuoco a, G. Gimelli a a b
Laboratorio di Citogenetica, Istituto Giannina Gaslini, Genova, Italy Clinica Pediatrica, Istituto Giannina Gaslini, Genova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 July 2014 Accepted 23 December 2014 Available online 14 January 2015
The 16p13.3p13.1 region has been reported as a “critical” hotspot region for recurrent microdeletions/ duplications, which may contribute to epilepsy, learning difficulties and facial dysmorphisms. Cytogenetic and array-CGH analyses were performed because of the clinical characteristics of the patient. The girl showed de novo 16p13.3p13.13 duplication spanning a region of w5.3 Mb. She presented brain anomalies, intellectual disability, epilepsy, facial and vertebral dysmorphisms. To our knowledge, this is the first reported case of 16p13.3p13.13 duplication; only three patients with an overlapping deletion in 16p13.2p13.13 were previously described. The duplicated region contains 21 OMIM genes and, six of them (RBFOX1, TMEM114, ABAT, PMM2, GRIN2A and, LITAF) were found to be associated with known diseases. Although no duplication of these genes has been described in the literature, we discuss here if they had some role in determining phenotype of our patient. Ó 2015 Elsevier Masson SAS. All rights reserved.
Keywords: 16p13.3p13.13 duplication Array-CGH
1. Introduction Partial trisomy 16p is a rare chromosome imbalance characterized by intellectual disability, prenatal and postnatal growth deficiency, facial anomalies, cleft palate, congenital heart defect, and urogenital anomalies. Only few cases were analysed by array-CGH and a number of reported cases had a concomitant anomaly of a second chromosome [Digilio et al., 2009]. The short arm of chromosome 16 is characterized by the presence of regions rich in Low Copy Repeats (LCRs) and segmental duplications that might predispose to non-allelic homologous recombination (NAHR). The 16p13.3p13.1 has been reported as a “critical” hotspot region for recurrent microdeletions/duplications [Digilio et al., 2009]. Deletions at the 16p13.11 locus have been described in individuals with schizophrenia [Ingason et al., 2011], autism [Ullmann et al., 2007], epilepsy [Heinzen et al., 2010; de Kovel et al., 2010; Mefford et al., 2010], attention deficit hyperactivity disorders [Williams et al., 2010], intellectual disability, microcephaly, and/or
* Corresponding author. Laboratorio di Citogenetica, Istituto G.Gaslini, L.go G.Gaslini 5, 16147 Genova, Italy. Tel.: þ39 (10) 5636922. E-mail address: [email protected] (E. Tassano). http://dx.doi.org/10.1016/j.ejmg.2014.12.016 1769-7212/Ó 2015 Elsevier Masson SAS. All rights reserved.
multiple congenital anomalies [Hannes et al., 2009; Ullmann et al., 2007]. Patients with reciprocal duplication have varied clinical features including behavioural abnormalities, cognitive impairment, congenital heart defects and skeletal manifestation, such as hypermobility, craniosynostosis and polydactyly [Ramalingam et al., 2011; Tropeano et al., 2013]. The variety of phenotypes and the presence of the rearrangement in unaffected relatives may be due to several factors, such as incomplete penetrance, variable expressivity and failure to recognize subtle manifestations, imprinting, and environmental factors. The same holds true for duplications of 16p13.3, comprising the CREBBP gene [Thienpont et al., 2010]. Reutlinger et al. [2010] reported on the first three patients with 16p13.2p13.13 deletion encompassing GRIN2A. They presented intellectual disability, various dysmorphisms, and epilepsy. Reciprocal duplications have never been reported. Here we report on the clinical and molecular characterization of previously undescribed de novo duplication at 16p13.3p13.13 located between the known region of 16p13.3 duplications, comprising CREBBP, and the recurrent 16p13.11 microdeletion/ duplication syndrome. The patient presented epilepsy, corpus callosum agenesis, coloboma, intellectual disability and facial and vertebral dysmorphisms.
E. Tassano et al. / European Journal of Medical Genetics 58 (2015) 194e198
2. Clinical report The patient is the second child of healthy and unrelated parents. Family history is unremarkable. During the mother’s pregnancy, an ultrasound examination showed ventriculomegaly of the third and lateral ventricles and agenesis of the corpus callosum. The patient was born at 39 weeks of gestation. She was small for gestational age: weight was 2439 g (2 SD), length 43.5 cm (3 SD) and head circumference was lower than normal (2 SD). APGAR score was normal (1’: 9, 5’: 9). The newborn had normal left eye, but the right one was characterized by macrophtalmia with convergent strabismus and eyelid ptosis. Fundoscopy revealed a big right iris and chorioretinal coloboma associated with severe visual impairment. A dermoid cyst was present at the top of the nose. On the 6th day of life dystonic movements with brisk reflexes were reported. Brain CT and MRI, performed during the first days of life, confirmed the presence of ventriculomegaly, complete agenesis of corpus callosum and hippocampal commissure with preservation of the anterior commissure, and highlighted a condition of colpocephaly and a frontonasal calcification (Fig. 1C). Subsequently, backbone X-ray and medulla MRI revealed butterfly vertebrae D11, D9, and in particular D10 (Fig. 1B). Abdominal ultrasound examination was normal. Since the early years of life, she has presented mild neurodevelopmental, speech and walking delay. She was able to walk at the age of twenty-four months and to speak her first words at the age of sixteen months. Sphincter control was delayed too. For this reason, she underwent psychomotor rehabilitation. Moreover, she had an episode of cyanosis and generalized hypotonia with spontaneous resolution. EEG was normal. At the age of six years eight months, psychometric tests
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were performed and yielded the following results: mental age was 3 years 6 months according to Goodenough graphic test and lower than 4 years according to Hilda Santucci test; total IQ was 53, verbal IQ 68, performance IQ 51, processing speed 52 (WPPSI III test). At seven years of age, she had a seizure episode of short duration, characterized by head and trunk deviation to the left. Psychomotor development was further slowing-down, with poor reactivity to external stimuli. EEG showed moderate abnormalities in the right parietaltemporal-occipital and mild in left occipital regions. Age dependent partial seizure with features of late onset benign childhood occipital epilepsy was diagnosed. The patient started antiepileptic drug with valproate, since then the seizures have been well controlled, no other episodes have happened. Today, at the age of 10 years and 6 months her height is 136,8 cm (25th percentile according to Tanner et al.), her weight is 49 kg (97th percentile according to Tanner et al.) and BMI is 26,2 kg/m2, corresponding to obesity of second degree according to RollandCachera references, and in particular she presented an important weight gain, due to hyperphagia from the age of three years, associated with facial dysmorphisms (bilateral ptosis, beaked nose, small low-set ears) and mild to moderate neurodevelopmental delay with tendency to peer aggression and attention deficit (Fig. 1A). 3. Materials and methods Standard GTG banding was performed at a resolution of 400e 550 bands on metaphase chromosomes from peripheral blood lymphocytes of the patient and her parents. Molecular karyotyping was performed on the girl and her parents using Human Genome
Fig. 1. A) Present clinical features: obesity and facial dysmorphisms, in particular bilateral ptosis, beaked nose (dermoid cyst of the nose tip was previously surgically removed), small low-set ears. B) Backbone X-ray: the arrow shows dysmorphic D9, D10, and D11 butterfly vertebrae. C) Brain MRI: sagittal T1-weighted image shows complete agenesis of corpus callosum (arrow) and hippocampal commissure with preservation of the anterior and a frontonasal calcification (arrowhead).
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E. Tassano et al. / European Journal of Medical Genetics 58 (2015) 194e198
CGH Microarray Kit G3 180 (Agilent Technologies, Palo Alto, USA) with w13 kb overall median probe spacing. Labelling and hybridization were performed following the protocols provided by the manufacturers. A graphical overview was obtained using the Agilent Genomic Workbench Lite Edition Software 6.5.0.18. FISH was performed using SureFISH 16p13.3 RBFOX1 (Green) (Agilent) and subtelomeric probe 16q (Orange) (Cytocell Aquarius, Cambridge, UK). 4. Results Cytogenetic analysis, performed on GTG-banded metaphases from cultured lymphocytes of the patient and his parents, showed normal karyotypes. Considering the phenotypic abnormalities of the patient, array-CGH analysis was performed, showing a 5.291 Mb de novo interstitial duplication at 16p13.3p13.13 bands. The duplication spanned from probe A_14_P200851 (6,754,986 bps) to probe A_16_P03116184 (12,046,383 bps) flanked by probe A_14_P134774 (6,754,720 bps) and probe A_14_P110158 (12,061,688 bps) (GRCh37/Hg19 assembly) (Fig. 2). No other additional CNVs were detected. FISH results confirmed the duplication on 16p13.3 of the proband and find no insertion translocations in her parents. The duplicated region contains 21 OMIM genes and, six of them (RBFOX1, TMEM114, ABAT, PMM2, GRIN2A and, LITAF) were found to be associated with known diseases (Supplementary Table 1). 5. Discussion In the present study, we describe a patient with a de novo 16p13.3p13.13 duplication presenting brain anomalies, intellectual disability, epilepsy, facial and vertebral dysmorphisms.
To our knowledge, this is the first reported case of 16p13.3p13.13 duplication; only three patients with an overlapping deletion in 16p13.2p13.13 were previously described [Reutlinger et al., 2010]. The duplication identified in our patient encompasses a region that is not reported as a copy number variant in the DGV (http:// projects.tcag.ca/variation/). This duplicated region has high OMIM gene content whose simultaneous presence makes very difficult to identify genotypeephenotype correlations and impossible to recognize the syndrome on clinical grounds. Moreover, to our knowledge, an overlapping duplication of comparable size has not been reported in the literature nor in publicly available databases DECIPHER (http://decipher.sanger.ac.uk) and ISCA (http://iscaconsortium.org). There are multiple smaller duplications on Decipher (n.268461, 267094, 291842, 251319, 267671, 278605). We contacted the authors to obtain the clinical informations, but we have had only general data (Supplementary Table 2). In generally they had psychomotor and intellectual disability, language delay and facial dysmorphisms. Interestingly, our patient shared a number of clinical features with some patients carrying the 16p13.2p13.13 deletion reported by Reutlinger et al. [2010]. These features included epilepsy, intellectual disability, delayed speech and motor development, and brain anomalies. However, our patient also presented some phenotypic features that were not present in the cases reported by Reutlinger et al. [2010], as macrophthalmia, coloboma, agenesis of corpus callosum and hippocampal commissure, colpocephaly and frontonasal calcification (Table 1). The genomic 16p13.3p13.13 duplicated region present in our patient contains 21 OMIM genes, two of which (GRIN2A and ATF7IP2) are common to all three microdeleted patients. Additionally, the region contains haploinsufficient (dosage sensitive)
Fig. 2. Overview of the region 16p13.3p13.13 and its gene content according to the UCSC Genome Browser [GRCh37/hg19 assembly]. The bars indicate the duplicated region (blue) in our patient and the deleted regions (red) in patients 1e3 and Decipher patients.
E. Tassano et al. / European Journal of Medical Genetics 58 (2015) 194e198
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Table 1 Comparison between phenotypic features of patients with deletions and duplication spanning GRIN2A.
Epilepsy EEG findings
Intellectual disability Language Motor developmental
Patient 1 Reutlinger et al., 2010
Patient 2 Reutlinger et al., 2010
Patient 3 Reutlinger et al., 2010
Our patient
þ Bilateral independent centrotemporal spikes, electrical status epilepticus in sleep (ESES) Severe Absent, mainly nonverbal communication Aided walking
þ Not available
þ Bilateral independent centrotemporal spikes reminiscent of ESES
Mild to moderate Delayed speech development
Severe Absent
þ Mild abnormalities in the right parieto-temporal-occipital region and the left occipital side. Mild to moderate Delayed speech developmental
Walking since age 2
No walking, able to sit and roll over Deep-set eyes, short nose with a broad nasal root, thin upper lip red, slight brachydactyly with tapering
Walking delay
e
Macroftalmia with convergent strabismus and eyelid ptosis. Big right iris and chorioretinic coloboma associated with severe visual impairment. Dermoid cyst at the top of the nose. Ventriculomegaly, complete agenesis of the corpus callosum and hippocampal commissure with preservation of the anterior commissure, colpocephaly, frontonasal calcification Dup(16) (p13.3p13.13) chr16:6,754,986e12,046,383
Low-set ears, epicanthus, mild hypertelorism, widow’s peak, low-set backward rotated ears with overfolded helices and poorly formed anthelices, brachydactyly, clinodactyly Cryptorchidism, choanal stenosis
Small and long face, mildly downslanting palpebral fissures, small narrow eyelids, broad nasal tip, long ears with large ear lobules, broad gums, broad neck, truncal, obesity Club feet, cryptorchidism
MR/CT
Minor periventricular leukomalacia in the posterior horns
CT scan unremarkable
Mild diffuse reduction of brain volume
Chromosome band Deleted region
Del(16)(p13.2p13.13) chr16:7,963,999e10,607,499 chr16:7,904,000e10,515,000 2.6 Mb
Del(16) (p13.2) chr16:8,992,499e9,992,499 chr16:8,900,000e9,900,000 1 Mb
Del(16) (p13.2p13.13) chr16:9,365,499e11,273,199 chr16:9,273,000e11,180,700 1.9 Mb
Dysmorphisms
Other malformation
Size
genes (PMM2, CARHSP1, USP7, NUBP1, TNP2) with haploinsufficiency (HI) score <50% [Huang et al., 2010]. The presence of dosage sensitive genes in the region supports the theory of overexpression phenotypes that resemble the underexpression ones [Conrad and Antonarakis, 2007]. This is an interesting hypothesis that could explain the common features of our patient and of those previously described with deletion in the same genomic region. Obviously, it is premature to define an ‘overexpression phenotype’ based on a sole observation of a single patient with a duplication, especially when the phenotype consists of epilepsy and intellectual disability, two common findings in patients with chromosomal imbalances. Nevertheless, in our opinion, this hypothesis remains suggestive. The same observations were made for 22q11.2 microduplication, complementary to 22q11.2 deletion causing DiGeorge syndrome, and for 16p13.3 microduplication, which is complementary to 16p13.3 microdeletion causing Rubinstein-Taybisyndrome [Portnoi et al., 2009; Thienpont et al., 2010]. Moreover, the defects observed in reciprocal deletion and duplication syndromes often involve the same organs or functions, for example, the heart in DiGeorge syndrome and 22q11.2duplication, speech in Williams-Beuren syndrome and 7q11.23 duplication, and the teeth in Smith-Magenis and PotockiLupski syndromes [Thienpont et al., 2010]. As regards epilepsy, that is a common feature to our patient and those reported by Reutlinger et al. [2010], we speculate that ABAT and GRIN2A may be possible candidate genes. In fact, the ABAT gene is involved in GABA metabolism and mutations of this gene have been reported in severe seizure disorders [Jaeken et al., 1984; Medina-Kauwe et al., 1999]. The GRIN2A gene encodes a subunit of the N-methyl-D-aspartate (NMDA) receptor NR2A that
Bilateral ptosis, beaked nose, small low-set years
5.291 Mb
mediates excitatory neurotransmission via glutamate. Mutations of this gene have been reported in individuals with focal epilepsy and speech disorders, with or without intellectual disability (MIM #245570) (summary by Carvill et al., 2013; Lemke et al., 2013; Lesca et al., 2013). However, as mentioned above, other anomalies characterize the phenotype of our patient. In fact, in this interval there are many other genes, five of them (MHC2TA, RBFOX1, PMM2, LITAF, BCAR4) with known disease associations. Their mutations cause autosomal recessive syndromes, while no abnormalities have been reported in duplications. However, we cannot exclude that the duplication of these or other genes might be responsible for the more severe phenotype and additional features of our patient. In conclusion, we described the first case of 16p13.3p13.13 duplication and compared the phenotype with that of the three patients described by Reutlinger et al. [2010] in order to highlight the main abnormalities. However, to further support our hypothesis, more cases with a similar novel microduplication need to be studied. Consent Written informed consent was obtained from the patient for publication of this paper and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.
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Authors’ contributions All authors have made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. All authors have been involved in drafting the manuscript and revising it critically for important intellectual content. All authors read and approved the final manuscript. Acknowledgements We thank the patient’s parents for their kind participation and support. We are grateful to Marco Bertorello and Corrado Torello for their technical assistance. This work was supported by “Cinque per mille dell’IRPEF- Finanziamento della ricerca sanitaria” and “Finanziamento Ricerca Corrente, Ministero Salute (contributo per la ricerca intramurale). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmg.2014.12.016. References Carvill GL, Regan BM, Yendle SC, et al. GRIN2A mutations cause epilepsy-aphasia spectrum disorders. Nat. Genet. 2013;45:1073e6. Conrad B, Antonarakis SE. Gene duplication: a drive for phenotypic diversity and cause of human disease. Annu. Rev. Genomics Hum. Genet. 2007;8:17e35. de Kovel CG, Trucks H, Helbig I, et al. Recurrent microdeletions at 15q11.2 and 16p13.11 predispose to idiopathic generalized epilepsies. Brain Jan. 2010;133:23e32. Digilio MC, Bernardini L, Capalbo A, et al. 16p subtelomeric duplication: a clinically recognizable syndrome. Eur. J. Hum. Genet. 2009;17:1135e40. Hannes FD, Sharp AJ, Mefford HC, et al. Recurrent reciprocal deletions and duplications of 16p13.11: the deletion is a risk factor for MR/MCA while the duplication may be a rare benign variant. J. Med. Genet. 2009;46:223e32.
Heinzen EL, Radtke RA, Urban TJ, et al. Rare deletions at 16p13.11 predispose to a diverse spectrum of sporadic epilepsy syndromes. Am. J. Hum. Genet. 2010;14: 707e18. Huang N, Lee I, Marcotte EM, et al. Characterising and predicting haploinsufficiency in the human genome. PLoS Genet. 2010;6:e1001154. Ingason A, Rujescu D, Cichon S, et al. Copy number variations of chromosome 16p13.1 region associated with schizophrenia. Mol. Psychiatr. 2011;16:17e 25. Jaeken J, Casaer P, de Cock P, et al. Gamma-aminobutyric acid-transaminase deficiency: a newly recognized inborn error of neurotransmitter metabolism. Neuropediatrics 1984;15:165e9. Lemke JR, Lal D, Reinthaler EM, et al. Mutations in GRIN2A cause idiopathic focal epilepsy with rolandic spikes. Nat. Genet. 2013;45:1067e72. Lesca G, Rudolf G, Bruneau N, et al. GRIN2A mutations in acquired epileptic aphasia and related childhood focal epilepsies and encephalopathies with speech and language dysfunction. Nat. Genet. 2013;45:1061e6. Medina-Kauwe LK, Tobin AJ, De Meirleir L, et al. 4-Aminobutyrate aminotransferase (GABA-transaminase) deficiency. J. Inherit. Metab. Dis. 1999;22:414e27. Mefford HC, Muhle H, Ostertag P, et al. Genome-wide copy number variation in epilepsy: novel susceptibility loci in idiopathic generalized and focal epilepsies. PLoS Genet. 2010;20:e1000962. Portnoi MF. Microduplication 22q11.2: a new chromosomal syndrome. Eur. J. Med. Genet. 2009;52:88e93. Ramalingam A, Zhou XG, Fiedler SD, et al. 16p13.11 duplication is a risk factor for a wide spectrum of neuropsychiatric disorders. J. Hum. Genet. 2011;56: 541e4. Reutlinger C, Helbig I, Gawelczyk B, et al. Deletions in 16p13 including GRIN2A in patients with intellectual disability, various dysmorphic features, and seizure disorders of the rolandic region. Epilepsia 2010;51:1870e3. Thienpont B, Béna F, Breckpot J, et al. Duplications of the critical Rubinstein-Taybi deletion region on chromosome 16p13.3 cause a novel recognisable syndrome. J. Med. Genet. 2010;47:155e61. Tropeano M, Ahn JW, Dobson RJ, et al. Male-biased autosomal effect of 16p13.11 copy number variation in neurodevelopmental disorders. PLoS One 2013;8: e61365. Ullmann R, Turner G, Kirchhoff M, et al. Array CGH identifies reciprocal 16p13.1 duplications and deletions that predispose to autism and/or mental retardation. Hum. Mutat. 2007;28:674e82. Williams NM, Zaharieva I, Martin A, et al. Rare chromosomal deletions and duplications in attention-deficit hyperactivity disorder: a genome-wide analysis. Lancet 2010;23:1401e8.
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Array report
Clinical and molecular delineation of a 16p13.2p13.13 microduplication E. Tassano a, *, M.G. Alpigiani b, A. Calcagno b, P. Salvati b, L. De Miglio b, P. Fiorio a, C. Cuoco a, G. Gimelli a a b
Laboratorio di Citogenetica, Istituto Giannina Gaslini, Genova, Italy Clinica Pediatrica, Istituto Giannina Gaslini, Genova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 July 2014 Accepted 23 December 2014 Available online 14 January 2015
The 16p13.3p13.1 region has been reported as a “critical” hotspot region for recurrent microdeletions/ duplications, which may contribute to epilepsy, learning difficulties and facial dysmorphisms. Cytogenetic and array-CGH analyses were performed because of the clinical characteristics of the patient. The girl showed de novo 16p13.3p13.13 duplication spanning a region of w5.3 Mb. She presented brain anomalies, intellectual disability, epilepsy, facial and vertebral dysmorphisms. To our knowledge, this is the first reported case of 16p13.3p13.13 duplication; only three patients with an overlapping deletion in 16p13.2p13.13 were previously described. The duplicated region contains 21 OMIM genes and, six of them (RBFOX1, TMEM114, ABAT, PMM2, GRIN2A and, LITAF) were found to be associated with known diseases. Although no duplication of these genes has been described in the literature, we discuss here if they had some role in determining phenotype of our patient. Ó 2015 Elsevier Masson SAS. All rights reserved.
Keywords: 16p13.3p13.13 duplication Array-CGH
1. Introduction Partial trisomy 16p is a rare chromosome imbalance characterized by intellectual disability, prenatal and postnatal growth deficiency, facial anomalies, cleft palate, congenital heart defect, and urogenital anomalies. Only few cases were analysed by array-CGH and a number of reported cases had a concomitant anomaly of a second chromosome [Digilio et al., 2009]. The short arm of chromosome 16 is characterized by the presence of regions rich in Low Copy Repeats (LCRs) and segmental duplications that might predispose to non-allelic homologous recombination (NAHR). The 16p13.3p13.1 has been reported as a “critical” hotspot region for recurrent microdeletions/duplications [Digilio et al., 2009]. Deletions at the 16p13.11 locus have been described in individuals with schizophrenia [Ingason et al., 2011], autism [Ullmann et al., 2007], epilepsy [Heinzen et al., 2010; de Kovel et al., 2010; Mefford et al., 2010], attention deficit hyperactivity disorders [Williams et al., 2010], intellectual disability, microcephaly, and/or
* Corresponding author. Laboratorio di Citogenetica, Istituto G.Gaslini, L.go G.Gaslini 5, 16147 Genova, Italy. Tel.: þ39 (10) 5636922. E-mail address: [email protected] (E. Tassano). http://dx.doi.org/10.1016/j.ejmg.2014.12.016 1769-7212/Ó 2015 Elsevier Masson SAS. All rights reserved.
multiple congenital anomalies [Hannes et al., 2009; Ullmann et al., 2007]. Patients with reciprocal duplication have varied clinical features including behavioural abnormalities, cognitive impairment, congenital heart defects and skeletal manifestation, such as hypermobility, craniosynostosis and polydactyly [Ramalingam et al., 2011; Tropeano et al., 2013]. The variety of phenotypes and the presence of the rearrangement in unaffected relatives may be due to several factors, such as incomplete penetrance, variable expressivity and failure to recognize subtle manifestations, imprinting, and environmental factors. The same holds true for duplications of 16p13.3, comprising the CREBBP gene [Thienpont et al., 2010]. Reutlinger et al. [2010] reported on the first three patients with 16p13.2p13.13 deletion encompassing GRIN2A. They presented intellectual disability, various dysmorphisms, and epilepsy. Reciprocal duplications have never been reported. Here we report on the clinical and molecular characterization of previously undescribed de novo duplication at 16p13.3p13.13 located between the known region of 16p13.3 duplications, comprising CREBBP, and the recurrent 16p13.11 microdeletion/ duplication syndrome. The patient presented epilepsy, corpus callosum agenesis, coloboma, intellectual disability and facial and vertebral dysmorphisms.
E. Tassano et al. / European Journal of Medical Genetics 58 (2015) 194e198
2. Clinical report The patient is the second child of healthy and unrelated parents. Family history is unremarkable. During the mother’s pregnancy, an ultrasound examination showed ventriculomegaly of the third and lateral ventricles and agenesis of the corpus callosum. The patient was born at 39 weeks of gestation. She was small for gestational age: weight was 2439 g (2 SD), length 43.5 cm (3 SD) and head circumference was lower than normal (2 SD). APGAR score was normal (1’: 9, 5’: 9). The newborn had normal left eye, but the right one was characterized by macrophtalmia with convergent strabismus and eyelid ptosis. Fundoscopy revealed a big right iris and chorioretinal coloboma associated with severe visual impairment. A dermoid cyst was present at the top of the nose. On the 6th day of life dystonic movements with brisk reflexes were reported. Brain CT and MRI, performed during the first days of life, confirmed the presence of ventriculomegaly, complete agenesis of corpus callosum and hippocampal commissure with preservation of the anterior commissure, and highlighted a condition of colpocephaly and a frontonasal calcification (Fig. 1C). Subsequently, backbone X-ray and medulla MRI revealed butterfly vertebrae D11, D9, and in particular D10 (Fig. 1B). Abdominal ultrasound examination was normal. Since the early years of life, she has presented mild neurodevelopmental, speech and walking delay. She was able to walk at the age of twenty-four months and to speak her first words at the age of sixteen months. Sphincter control was delayed too. For this reason, she underwent psychomotor rehabilitation. Moreover, she had an episode of cyanosis and generalized hypotonia with spontaneous resolution. EEG was normal. At the age of six years eight months, psychometric tests
195
were performed and yielded the following results: mental age was 3 years 6 months according to Goodenough graphic test and lower than 4 years according to Hilda Santucci test; total IQ was 53, verbal IQ 68, performance IQ 51, processing speed 52 (WPPSI III test). At seven years of age, she had a seizure episode of short duration, characterized by head and trunk deviation to the left. Psychomotor development was further slowing-down, with poor reactivity to external stimuli. EEG showed moderate abnormalities in the right parietaltemporal-occipital and mild in left occipital regions. Age dependent partial seizure with features of late onset benign childhood occipital epilepsy was diagnosed. The patient started antiepileptic drug with valproate, since then the seizures have been well controlled, no other episodes have happened. Today, at the age of 10 years and 6 months her height is 136,8 cm (25th percentile according to Tanner et al.), her weight is 49 kg (97th percentile according to Tanner et al.) and BMI is 26,2 kg/m2, corresponding to obesity of second degree according to RollandCachera references, and in particular she presented an important weight gain, due to hyperphagia from the age of three years, associated with facial dysmorphisms (bilateral ptosis, beaked nose, small low-set ears) and mild to moderate neurodevelopmental delay with tendency to peer aggression and attention deficit (Fig. 1A). 3. Materials and methods Standard GTG banding was performed at a resolution of 400e 550 bands on metaphase chromosomes from peripheral blood lymphocytes of the patient and her parents. Molecular karyotyping was performed on the girl and her parents using Human Genome
Fig. 1. A) Present clinical features: obesity and facial dysmorphisms, in particular bilateral ptosis, beaked nose (dermoid cyst of the nose tip was previously surgically removed), small low-set ears. B) Backbone X-ray: the arrow shows dysmorphic D9, D10, and D11 butterfly vertebrae. C) Brain MRI: sagittal T1-weighted image shows complete agenesis of corpus callosum (arrow) and hippocampal commissure with preservation of the anterior and a frontonasal calcification (arrowhead).
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E. Tassano et al. / European Journal of Medical Genetics 58 (2015) 194e198
CGH Microarray Kit G3 180 (Agilent Technologies, Palo Alto, USA) with w13 kb overall median probe spacing. Labelling and hybridization were performed following the protocols provided by the manufacturers. A graphical overview was obtained using the Agilent Genomic Workbench Lite Edition Software 6.5.0.18. FISH was performed using SureFISH 16p13.3 RBFOX1 (Green) (Agilent) and subtelomeric probe 16q (Orange) (Cytocell Aquarius, Cambridge, UK). 4. Results Cytogenetic analysis, performed on GTG-banded metaphases from cultured lymphocytes of the patient and his parents, showed normal karyotypes. Considering the phenotypic abnormalities of the patient, array-CGH analysis was performed, showing a 5.291 Mb de novo interstitial duplication at 16p13.3p13.13 bands. The duplication spanned from probe A_14_P200851 (6,754,986 bps) to probe A_16_P03116184 (12,046,383 bps) flanked by probe A_14_P134774 (6,754,720 bps) and probe A_14_P110158 (12,061,688 bps) (GRCh37/Hg19 assembly) (Fig. 2). No other additional CNVs were detected. FISH results confirmed the duplication on 16p13.3 of the proband and find no insertion translocations in her parents. The duplicated region contains 21 OMIM genes and, six of them (RBFOX1, TMEM114, ABAT, PMM2, GRIN2A and, LITAF) were found to be associated with known diseases (Supplementary Table 1). 5. Discussion In the present study, we describe a patient with a de novo 16p13.3p13.13 duplication presenting brain anomalies, intellectual disability, epilepsy, facial and vertebral dysmorphisms.
To our knowledge, this is the first reported case of 16p13.3p13.13 duplication; only three patients with an overlapping deletion in 16p13.2p13.13 were previously described [Reutlinger et al., 2010]. The duplication identified in our patient encompasses a region that is not reported as a copy number variant in the DGV (http:// projects.tcag.ca/variation/). This duplicated region has high OMIM gene content whose simultaneous presence makes very difficult to identify genotypeephenotype correlations and impossible to recognize the syndrome on clinical grounds. Moreover, to our knowledge, an overlapping duplication of comparable size has not been reported in the literature nor in publicly available databases DECIPHER (http://decipher.sanger.ac.uk) and ISCA (http://iscaconsortium.org). There are multiple smaller duplications on Decipher (n.268461, 267094, 291842, 251319, 267671, 278605). We contacted the authors to obtain the clinical informations, but we have had only general data (Supplementary Table 2). In generally they had psychomotor and intellectual disability, language delay and facial dysmorphisms. Interestingly, our patient shared a number of clinical features with some patients carrying the 16p13.2p13.13 deletion reported by Reutlinger et al. [2010]. These features included epilepsy, intellectual disability, delayed speech and motor development, and brain anomalies. However, our patient also presented some phenotypic features that were not present in the cases reported by Reutlinger et al. [2010], as macrophthalmia, coloboma, agenesis of corpus callosum and hippocampal commissure, colpocephaly and frontonasal calcification (Table 1). The genomic 16p13.3p13.13 duplicated region present in our patient contains 21 OMIM genes, two of which (GRIN2A and ATF7IP2) are common to all three microdeleted patients. Additionally, the region contains haploinsufficient (dosage sensitive)
Fig. 2. Overview of the region 16p13.3p13.13 and its gene content according to the UCSC Genome Browser [GRCh37/hg19 assembly]. The bars indicate the duplicated region (blue) in our patient and the deleted regions (red) in patients 1e3 and Decipher patients.
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Table 1 Comparison between phenotypic features of patients with deletions and duplication spanning GRIN2A.
Epilepsy EEG findings
Intellectual disability Language Motor developmental
Patient 1 Reutlinger et al., 2010
Patient 2 Reutlinger et al., 2010
Patient 3 Reutlinger et al., 2010
Our patient
þ Bilateral independent centrotemporal spikes, electrical status epilepticus in sleep (ESES) Severe Absent, mainly nonverbal communication Aided walking
þ Not available
þ Bilateral independent centrotemporal spikes reminiscent of ESES
Mild to moderate Delayed speech development
Severe Absent
þ Mild abnormalities in the right parieto-temporal-occipital region and the left occipital side. Mild to moderate Delayed speech developmental
Walking since age 2
No walking, able to sit and roll over Deep-set eyes, short nose with a broad nasal root, thin upper lip red, slight brachydactyly with tapering
Walking delay
e
Macroftalmia with convergent strabismus and eyelid ptosis. Big right iris and chorioretinic coloboma associated with severe visual impairment. Dermoid cyst at the top of the nose. Ventriculomegaly, complete agenesis of the corpus callosum and hippocampal commissure with preservation of the anterior commissure, colpocephaly, frontonasal calcification Dup(16) (p13.3p13.13) chr16:6,754,986e12,046,383
Low-set ears, epicanthus, mild hypertelorism, widow’s peak, low-set backward rotated ears with overfolded helices and poorly formed anthelices, brachydactyly, clinodactyly Cryptorchidism, choanal stenosis
Small and long face, mildly downslanting palpebral fissures, small narrow eyelids, broad nasal tip, long ears with large ear lobules, broad gums, broad neck, truncal, obesity Club feet, cryptorchidism
MR/CT
Minor periventricular leukomalacia in the posterior horns
CT scan unremarkable
Mild diffuse reduction of brain volume
Chromosome band Deleted region
Del(16)(p13.2p13.13) chr16:7,963,999e10,607,499 chr16:7,904,000e10,515,000 2.6 Mb
Del(16) (p13.2) chr16:8,992,499e9,992,499 chr16:8,900,000e9,900,000 1 Mb
Del(16) (p13.2p13.13) chr16:9,365,499e11,273,199 chr16:9,273,000e11,180,700 1.9 Mb
Dysmorphisms
Other malformation
Size
genes (PMM2, CARHSP1, USP7, NUBP1, TNP2) with haploinsufficiency (HI) score <50% [Huang et al., 2010]. The presence of dosage sensitive genes in the region supports the theory of overexpression phenotypes that resemble the underexpression ones [Conrad and Antonarakis, 2007]. This is an interesting hypothesis that could explain the common features of our patient and of those previously described with deletion in the same genomic region. Obviously, it is premature to define an ‘overexpression phenotype’ based on a sole observation of a single patient with a duplication, especially when the phenotype consists of epilepsy and intellectual disability, two common findings in patients with chromosomal imbalances. Nevertheless, in our opinion, this hypothesis remains suggestive. The same observations were made for 22q11.2 microduplication, complementary to 22q11.2 deletion causing DiGeorge syndrome, and for 16p13.3 microduplication, which is complementary to 16p13.3 microdeletion causing Rubinstein-Taybisyndrome [Portnoi et al., 2009; Thienpont et al., 2010]. Moreover, the defects observed in reciprocal deletion and duplication syndromes often involve the same organs or functions, for example, the heart in DiGeorge syndrome and 22q11.2duplication, speech in Williams-Beuren syndrome and 7q11.23 duplication, and the teeth in Smith-Magenis and PotockiLupski syndromes [Thienpont et al., 2010]. As regards epilepsy, that is a common feature to our patient and those reported by Reutlinger et al. [2010], we speculate that ABAT and GRIN2A may be possible candidate genes. In fact, the ABAT gene is involved in GABA metabolism and mutations of this gene have been reported in severe seizure disorders [Jaeken et al., 1984; Medina-Kauwe et al., 1999]. The GRIN2A gene encodes a subunit of the N-methyl-D-aspartate (NMDA) receptor NR2A that
Bilateral ptosis, beaked nose, small low-set years
5.291 Mb
mediates excitatory neurotransmission via glutamate. Mutations of this gene have been reported in individuals with focal epilepsy and speech disorders, with or without intellectual disability (MIM #245570) (summary by Carvill et al., 2013; Lemke et al., 2013; Lesca et al., 2013). However, as mentioned above, other anomalies characterize the phenotype of our patient. In fact, in this interval there are many other genes, five of them (MHC2TA, RBFOX1, PMM2, LITAF, BCAR4) with known disease associations. Their mutations cause autosomal recessive syndromes, while no abnormalities have been reported in duplications. However, we cannot exclude that the duplication of these or other genes might be responsible for the more severe phenotype and additional features of our patient. In conclusion, we described the first case of 16p13.3p13.13 duplication and compared the phenotype with that of the three patients described by Reutlinger et al. [2010] in order to highlight the main abnormalities. However, to further support our hypothesis, more cases with a similar novel microduplication need to be studied. Consent Written informed consent was obtained from the patient for publication of this paper and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.
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Authors’ contributions All authors have made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. All authors have been involved in drafting the manuscript and revising it critically for important intellectual content. All authors read and approved the final manuscript. Acknowledgements We thank the patient’s parents for their kind participation and support. We are grateful to Marco Bertorello and Corrado Torello for their technical assistance. This work was supported by “Cinque per mille dell’IRPEF- Finanziamento della ricerca sanitaria” and “Finanziamento Ricerca Corrente, Ministero Salute (contributo per la ricerca intramurale). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmg.2014.12.016. References Carvill GL, Regan BM, Yendle SC, et al. GRIN2A mutations cause epilepsy-aphasia spectrum disorders. Nat. Genet. 2013;45:1073e6. Conrad B, Antonarakis SE. Gene duplication: a drive for phenotypic diversity and cause of human disease. Annu. Rev. Genomics Hum. Genet. 2007;8:17e35. de Kovel CG, Trucks H, Helbig I, et al. Recurrent microdeletions at 15q11.2 and 16p13.11 predispose to idiopathic generalized epilepsies. Brain Jan. 2010;133:23e32. Digilio MC, Bernardini L, Capalbo A, et al. 16p subtelomeric duplication: a clinically recognizable syndrome. Eur. J. Hum. Genet. 2009;17:1135e40. Hannes FD, Sharp AJ, Mefford HC, et al. Recurrent reciprocal deletions and duplications of 16p13.11: the deletion is a risk factor for MR/MCA while the duplication may be a rare benign variant. J. Med. Genet. 2009;46:223e32.
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