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16p13 microduplication without CREBBP involvement: Moving toward a phenotype delineation
European Journal of Medical Genetics xxx (2016) 1e4
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
16p13 microduplication without CREBBP involvement: Moving toward a phenotype delineation Claudia Ciaccio a, *, Arianna Tucci a, Giulietta Scuvera a, Margherita Estienne b, Susanna Esposito a, Donatella Milani a degli Studi di Milano, Fondazione IRCCS Ca' Granda Pediatric Highly Intensive Care Unit, Department of Pathophysiology and Transplantation, Universita Ospedale Maggiore Policlinico, Via Commenda 9, 20122, Milan, Italy b Foundation IRCCS Neurological Institute Carlo Besta, Milan, Italy a
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 June 2016 Received in revised form 12 December 2016 Accepted 16 December 2016 Available online xxx
The short arm of chromosome 16 is one of the less stable regions of our genome, as over 10% of the euchromatic region of 16p is composed of highly complex low copy repeats that are known to be predisposed to rearrangements mediated by non-allelic homologous recombination. The 16p13.3p13.13 molecular region has been defined as the 16p duplication hotspot, and duplications of chromosome 16p13 have recently been confirmed to cause a recognizable syndrome, with CREBBP being the main phenotype-causing gene. To date, only one case report is present in the literature with a 16p13 duplication without CREBBP involvement; we describe here a second analogous case with a not previously reported 16p13.2p13.13 microduplication. This paper allows us to better delineate the clinical features of 16p13 microduplications that do not encompass CREBBP and, concurrently, to narrow the molecular region responsible for congenital heart defects in 16p duplications as well as to propose GRIN2A as a candidate gene for epilepsy. © 2016 Elsevier Masson SAS. All rights reserved.
Keywords: 16p13 microduplication Genotype-phenotype correlation GRIN2A Epilepsy
1. Introduction Partial trisomy 16p is a rare chromosome imbalance that has been well described in the literature. This condition is characterized by global developmental delay (DD), intellectual disability (ID), facial dysmorphisms, and congenital anomalies that can affect any apparatus. The mortality rate in infancy is approximately 50%, but rare long-term survivors have been reported (Martin et al., 2002). The short arm of chromosome 16 is rich in low copy repeats (LCRs), which are known to predispose to rearrangements mediated by non-allelic homologous recombination (NAHR) of the molecular regions they flank; over 10% of the euchromatic region of the short arm of chromosome 16 is in fact composed of highly complex LCRs (Martin et al., 2004). In particular, an unusual and complex class of segmental duplications has been identified whose distribution is, for the most part, specific to the short arm of
* Corresponding author. Pediatric Highly Intensive Care Unit, Department of Pathophysiology and Transplantation, Universit a degli Studi di Milano, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Via della Commenda 9, 20122 Milano, Italy. E-mail address: [email protected] (C. Ciaccio).
chromosome 16 (Stallings et al., 1992). The most prolific chromosome-specific duplication on chromosome 16, termed LCR16a, is a 20 Kb duplication distributed in a non-tandem fashion to multiple locations along the entire length of chromosome 16, including cytogenetic band positions 16q22.2, 16q23, 16p11, 16p12.2, 16p12.3, 16p13.1, and 16p13.3 (Eichler et al., 2011). This duplication may potentially lead to NAHR in more than one region of the short arm of chromosome 16, explaining the high variability of breakpoints and size of the 16p microdeletions and microduplications. The most frequently implicated region in small chromosome rearrangements, defined as the 16p duplication hotspot, is the 16p13.3p13.13 segment (Digilio et al., 2009; Martin et al., 2002). Patients with a duplication of the critical 16p13.3p13.13 molecular region show clinical features that are similar to those shown by patients carrying bigger duplications (Digilio et al., 2009; Tassano et al., 2015). Recurrent clinical findings in the patients affected by this condition are mild to moderate ID, pre- and postnatal growth retardation, hypotonia, and speech and motor delay (language can also be absent). Facial dysmorphisms are distinct and some of them seem to follow an inverted pattern when compared to those shown by individuals with the opposite molecular defect (16p13.3 deletion) (Hammond et al., 2014). Patients
http://dx.doi.org/10.1016/j.ejmg.2016.12.006 1769-7212/© 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Ciaccio, C., et al., 16p13 microduplication without CREBBP involvement: Moving toward a phenotype delineation, European Journal of Medical Genetics (2016), http://dx.doi.org/10.1016/j.ejmg.2016.12.006
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can show microcephaly, ear anomalies, hypertelorism, upslanting and narrow palpebral fissures, prominent glabella, flat nasal bridge, short nose, anteverted nares, flat philtrum, cleft palate, thin lips and micrognathia. The neurological findings include autism spectrum disorder, attention deficit/hyperactivity disorder (ADHD), epilepsy, and brain MRI abnormalities. Any apparatus can potentially be involved: cardiovascular problems (i.e., septal defects, patent ductus arteriosus, mitral valve prolapse, pulmonary insufficiency/stenosis/hypertension), genito-urinary malformations (i.e., renal hypoplasia, vesicourethral reflux, cryptorchidism, small penis, shawl scrotum), skeletal anomalies (i.e., scoliosis, winged scapulae, pectus excavatum, metacarpal bones and phalanx hypoplasia, small hands and feet, camptodactyly, arthrogryposis), refraction defects, and macroglossia have all been reported. This latter aspect, in association with facial muscle hypotonia, can explain the typical tendency to hold the mouth open. Less common findings include inguinal or umbilical hernia, irides dyschromia, dental anomalies (crowding or small teeth), joint hyperlaxity, and different minor skeletal anomalies that are rarely observed (Demeer et al., 2013; Digilio et al., 2009; Li et al., 2013; Marangi et al., 2008; Martin et al., 2002; Mattina et al., 2012; Rochat et al., 2007; Thienpont et al., 2010). All the 16p13.3p13.13 duplications described thus far, except for one, include the CREBBP gene (chromosome 16:3,775,0563,930,121), whose deletion or mutation is responsible for Rubinstein-Taybi Syndrome type 1 (RSTS, OMIM #180849). Duplications of chromosome 16p13.3 encompassing CREBBP have therefore been proposed and later confirmed to cause a recognizable syndrome, with CREBBP being the main gene (Demeer et al., 2013; Marangi et al., 2008; Mattina et al., 2012; Thienpont et al., 2010). The prevalence of this rearrangement has been estimated to account for around 0.043% of all cases of DD (Thienpont et al., 2010). To date, only one patient has been described carrying a 16p13.3p13.13 duplication without CREBBP involvement (Tassano et al., 2015). The patient shares most of the clinical attributes reported in patients carrying duplications in the classical 16p13.3p13.13 hotspot, though the phenotype is milder. Here, we report the case of a girl referred to our Center for a pathological Array-CGH analysis who was found to have a 16p13.2p13.13 microduplication. This report allows us to better delineate the clinical features of 16p13 microduplications not encompassing CREBBP and, concurrently, to narrow the molecular region responsible for congenital heart defects in patients with 16p duplications as well as to propose GRIN2A as a candidate gene for epilepsy.
abnormalities and an absence of epilepsy suggestive waves. An awake EEG performed a year later substantially confirmed the previous result, showing signal asymmetry and occasional slow waves in the anterior regions. Brain MRI (performed at 6 years) was normal. At the age of 7, the girl underwent a neuropsychiatric evaluation, confirming the diagnosis of ID and mood and behavior disorder. A full scale IQ test (e.g. WISC-V) cannot be performed because of the oppositional defiant behavior of the girl. She started anti-psychotic treatment with risperidone, but she never obtained full control of the symptoms. Genetic tests were performed: karyotype, telomere FISH analysis, and FMR1 molecular analysis were all negative. Clinical evaluation at 8 years old revealed growth parameters at the lower centiles of the normal range: height 121 cm (25th centile), weight 19,5 kg (3rd centile), height circumference 39 cm (3e10th centile). She showed a few non-specific facial dysmorphisms, including micrognathia, palpebral ptosis, sparse eyebrows, short philtrum, and thin superior lip (Fig. 1). Mild muscle hypotonia and hyperactivity were present; language was poor and incoherent. Peculiarly, she presented hypotonia and defective control of tongue and mouth muscles, leading to sialorrhea. The Array-CGH analysis was performed in an external laboratory, according to standard protocol, using an oligonucleotide-array with an average resolution of 180 Kb (assembly NCBI Build 36.1-hg 18, March 2006). The analysis showed a 1.63 Mb duplication at 16p13.2p13.13 (arr16p13.2p13.13 (9,598,196-11,231,095)x3) encompassing 6 known OMIM genes: GRIN2A (OMIM*138253), ATF7IP2 (OMIM*613645), EMP2 (OMIM*602334), NUBP1 (OMIM*600280), CIITA (OMIM*600005) and CLEC16A (OMIM*611303) (Fig. 2). qPCR was administered to confirm the cytogenetic finding, the Array-CGH analysis performed in the
2. Clinical report Our patient is an 8-year-old girl who came to our attention because of a pathological result from an Array-CGH analysis, which was performed for psychomotor delay and ID. She was the second child of healthy non-consanguineous parents; the family history was negative for ID and any other genetic conditions. She was born at term and did not experience perinatal distress; the neonatal parameters were within the normal range (length 51 cm, weight 3960 g, head circumference 35 cm). Her growth had always been normal for her height and weight. Mild psychomotor delay was present: she was able to walk independently and uttered her first words at 24 months. Inappropriate behavior had been present since first infancy, with attention deficit, mood disorder and generalized anxiety disorder (GAD). Moreover, she experienced absence epilepsy episodes, with a mean duration of 7e8 min and progressively increasing frequency. An EEG sleep analysis, performed at the age of 4, showed non-specific
Fig. 1. Facial appearance of the patient: low hairline, micrognathia, hypertelorism, mild palpebral ptosis, short philtrum, thin superior lip.
Please cite this article in press as: Ciaccio, C., et al., 16p13 microduplication without CREBBP involvement: Moving toward a phenotype delineation, European Journal of Medical Genetics (2016), http://dx.doi.org/10.1016/j.ejmg.2016.12.006
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Fig. 2. Map of genetic duplication in our patient and in patient previously described by Tassano et al. (2015). The results of the Array-CGH analysis have been uploaded to the UCSC Genome browser (GRCh37 Feb. 2009, hg 19).
parents was negative, confirming that the duplication occurred de novo. 3. Discussion The patient here described is the first case of 16p13.2p13.13 duplication that has ever been reported; it therefore allows us to better delineate the phenotype of 16p13 microduplication not involving CREBBP, condition that has to date been reported in only one patient, by Tassano et al. (2015). There are multiple duplications listed in Decipher with analogous (264947) or smaller dimensions (268461, 263932, 271072, 275724, 314649, and 289949). For most of those duplications, clinical data are not available. In general, they show psychomotor delay/ID, behavioral anomalies, microcephaly, and not better defined facial dysmorphisms. A clinical comparison between our patient and the patient reported by Tassano et al. (2015) is shown in Table 1, while a molecular comparison is shown in Fig. 2. The two patients share most of their features with patients carrying the classic 16p13.13p13.3 microduplication. DD is present in both cases, as well as ID, language delay, and hypotonia. This latter feature may contribute to the attitude of holding the mouth open that is exhibited by our patients. They show common facial dysmorphisms such as sparse eyebrows, palpebral ptosis, a short
philtrum, and a thin superior lip. Additional ocular abnormalities are present in both patients, but they seem to be of a non-specific fashion. Our patient presents hypertelorism (a feature that is also described in patients carrying the classical 16p13.13p13.3 duplication) and a lack of the inferomedial eyelashes (never previously reported); otherwise, the patient reported by Tassano et al. (2015) shows strabismus and right coloboma and macrophthalmia. Cardiovascular anomalies (septal defects, patent ductus arteriosus, mitral valve prolapse, pulmonary insufficiency/stenosis/hypertension) are well represented in patients with the classical 16p13.13p13.3 duplication or with bigger 16p13 duplications (Digilio et al., 2009; Li et al., 2013; Mattina et al., 2012; Rochat et al., 2007; Thienpont et al., 2010) but are absent in both current patients. We can therefore speculate that the region responsible for cardiovascular involvement falls outside the breakpoints of the duplication carried by patient 2. This finding may be of particular interest for the patients' prognosis, as idiopathic pulmonary hypertension appears to be the most severe complication of 16p distal duplications, and it has been previously reported as the primary cause of morbidity in a child carrying a 16p duplication (Movahhedian et al., 1998). The neuropsychiatric profile is similar and is characterized by attention deficit disorder and hyperphagia. In spite of that latter element, our patients' weight was at the 3rd centile, while patient 2 is overweight. Epilepsy is present in both
Table 1 Phenotypic comparison of patients carrying a subtelomeric 16p duplication.
Site (Hg19) and size of 16p duplication
Sex/age at evaluation ID GDD SGA Poor growth Hypotonia Micrognathia Ear anomalies Palpebral ptosis Sparse eyebrows Other ocular features Short philtrum Superior lip anomalies Epilepsy EEG anomalies Brain MRI anomalies Neuropsychiatric profile CV anomalies Skeletal anomalies
Patient 1 Present case
Patient 2 Tassano et al., 2015
arr16p13.2p13.13 (9,598,196-11,231,095)x3 1.63 Mb F/8 y þ þ þ þ þ þ þ Hypertelorism, absence of inferomedial eyelashes þ Thin vermilion Absence epilepsy Non-specific asymmetry Attention deficit, hyperactivity, behavior and mood disorder, GAD, hyperphagia
arr16p13.3p13.13 (6,754,986-12,046383)x3 5.3 Mb F/10.5 þ þ þ þ þ þ þ Right coloboma and macrophthalmia, strabismus þ Thin vermilion Late-onset benign childhood occipital epilepsy Moderate non-specific abnormalities after seizures episodes Ventriculomegaly, corpus callosum agenesis, colpocephaly Attention deficit, hetero-aggression, hyperphagia Butterfly D9-D10-D11
Abbreviations: Pt.: patient; GDD: global developmental delay; SGA: small for gestational age; GAD: generalized anxiety disorder; CV: cardiovascular.
Please cite this article in press as: Ciaccio, C., et al., 16p13 microduplication without CREBBP involvement: Moving toward a phenotype delineation, European Journal of Medical Genetics (2016), http://dx.doi.org/10.1016/j.ejmg.2016.12.006
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patients and is associated with non-specific EEG abnormalities; patient 2 also exhibits brain MRI anomalies (a common feature of the classical 16p13.13p13.3 microduplication). In both cases, the molecular region of the duplication includes the GRIN2A gene, which is not disrupted but is entirely duplicated. GRIN2A is a wellknown and widely studied gene as it encodes the alpha subunit of the ionotropic N-methyl-D-aspartate (NMDA) receptor; this ligandgated receptor is permeable to calcium ions, and its activation results in a calcium influx into post-synaptic cells, with the consequent trigger of several signaling cascades. The NMDA receptor determines key features of the synaptic response and has important consequences for synaptic plasticity and cell physiology (Kalia et al., 2008). In recent years, mutations of the GRIN2A gene have been associated with epilepsy, ID and language impairment (Lesca et al., 2013; Lemke et al., 2013). Lesca et al. (2013) identified a variety of GRIN2A mutations, mostly missense, as one of the major causes of familial epilepsy. Duplications of the gene encoding the alpha subunit of the NDMA receptor could potentially influence the final product at any level, altering the fine modulation of glutamate signaling. Supporting this hypothesis, both patients suffer from epilepsy, as reported for patients carrying GRIN2A mutations. In their paper, Tassano et al. already proposed GRIN2A or ABAT, both of which are within the 16p13 duplicated region of their patient, as possible causal genes for epilepsy. The duplication reported here does not encompass the ABAT gene; we can therefore infer that GRIN2A is the major gene implicated in the epileptic phenotype of these patients. In any case, the lack of functional studies about GRIN2A duplications preclude our going beyond this speculation. Further reports of patients carrying a 16p13 microduplication without CREBBP involvement are necessary to better delineate the phenotype and to confirm our cardiovascular and neurological data. Going forward, a better knowledge of this peculiar chromosomal region will be essential to establishing a patient-tailored follow-up, based also on the molecular defect. Acknowledgments The authors thank the patient and her parents for their cooperation during the diagnostic process. No funding was active on this study. All authors declare that there is no conflict of interest concerning this work. References Demeer, B., Andrieux, J., Receveur, A., Morin, G., Petit, F., Julia, S., Plessis, G., Martin€rs, K., Le Coignard, D., Delobel, B., Firth, H.V., Thuresson, A.C., Lanco Dosen, S., Sjo Caignec, C., Devriendt, K., Mathieu-Dramard, M., 2013. Duplication 16p13.3 and the CREBBP gene: confirmation of the phenotype. Eur. J. Med. Genet. 56 (1), 26e31. Digilio, M.C., Bernardini, L., Capalbo, A., Capolino, R., Gagliardi, M.G., Marino, B., Novelli, A., Dallapiccola, B., 2009. 16p subtelomeric duplication: a clinically recognizable syndrome. Eur. J. Hum. Genet. 17 (9), 1135e1140. Eichler, E.E., Johnson, M.E., Alkan, C., Tuzun, E., Sahinalp, C., Misceo, D., Archidiacono, N., Rocchi, M., 2011. Divergent origins and concerted expansion of two segmental duplications on chromosome 16. J. Hered. 92 (6), 462e468.
Hammond, P., McKee, S., Suttie, M., Allanson, J., Cobben, J.M., Maas, S.M., Quarrell, O., Smith, A.C., Lewis, S., Tassabehji, M., Sisodiya, S., Mattina, T., Hennekam, R., 2014. Opposite effects on facial morphology due to gene dosage sensitivity. Hum. Genet. 133 (9), 1117e1125. Kalia, L.V., Kalia, S.K., Salter, M.W., 2008. NMDA receptors in clinical neurology: excitatory times ahead. Lancet Neurol. 7 (8), 742e755. Lemke, J.R., Lal, D., Reinthaler, E.M., Steiner, I., Nothnagel, M., Alber, M., Geider, K., Laube, B., Schwake, M., Finsterwalder, K., Franke, A., Schilhabel, M., J€ ahn, J.A., Muhle, H., Boor, R., Van Paesschen, W., Caraballo, R., Fejerman, N., Weckhuysen, S., De Jonghe, P., Larsen, J., Møller, R.S., Hjalgrim, H., Addis, L., Tang, S., Hughes, E., Pal, D.K., Veri, K., Vaher, U., Talvik, T., Dimova, P., Guerrero pez, R., Serratosa, J.M., Linnankivi, T., Lehesjoki, A.E., Ruf, S., Wolff, M., Lo Buerki, S., Wohlrab, G., Kroell, J., Datta, A.N., Fiedler, B., Kurlemann, G., Kluger, G., Hahn, A., Haberlandt, D.E., Kutzer, C., Sperner, J., Becker, F., € ck, H., Neophythou, B., Ronen, G.M., GruberWeber, Y.G., Feucht, M., Steinbo Sedlmayr, U., Geldner, J., Harvey, R.J., Hoffmann, P., Herms, S., Altmüller, J., Toliat, M.R., Thiele, H., Nürnberg, P., Wilhelm, C., Stephani, U., Helbig, I., Lerche, H., Zimprich, F., Neubauer, B.A., Biskup, S., von Spiczak, S., 2013. Mutations in GRIN2A cause idiopathic focal epilepsy with rolandic spikes. Nat. Genet. 45, 1067e1072. Lesca, G., Rudolf, G., Bruneau, N., Lozovaya, N., Labalme, A., Boutry-Kryza, N., Salmi, M., Tsintsadze, T., Addis, L., Motte, J., Wright, S., Tsintsadze, V., Michel, A., Doummar, D., Lascelles, K., Strug, L., Waters, P., de Bellescize, J., Vrielynck, P., de Saint Martin, A., Ville, D., Ryvlin, P., Arzimanoglou, A., Hirsch, E., Vincent, A., Pal, D., Burnashev, N., Sanlaville, D., Szepetowski, P., 2013. GRIN2A mutations in acquired epileptic aphasia and related childhood focal epilepsies and encephalopathies with speech and language dysfunction. Nat. Genet. 45 (9), 1061e1066. Li, Z., Liu, J., Li, H., Peng, Y., Lv, W., Long, Z., Liang, D., Wu, L., 2013. Phenotypic expansion of the interstitial 16p13.3 duplication: a case report and review of the literature. Gene 531 (2), 502e505. Marangi, G., Leuzzi, V., Orteschi, D., Grimaldi, M.E., Lecce, R., Neri, G., Zollino, M., 2008. Duplication of the Rubinstein-Taybi region on 16p13.3 is associated with a distinctive phenotype. Am. J. Med. Genet. A 146A (18), 2313e2317. Martin, C.L., Waggoner, D.J., Wong, A., Uhrig, S., Roseberry, J.A., Hedrick, J.F., Pack, S.D., Russell, K., Zackai, E., Dobyns, W.B., Ledbetter, D.H., 2002. “Molecular rulers” for calibrating phenotypic effects of telomere imbalance. J. Med. Genet. 39 (10), 734e740. Martin, J., Han, C., Gordon, L.A., Terry, A., Prabhakar, S., She, X., et al., 2004. The sequence and analysis of duplication-rich human chromosome 16. Nature 432, 988e994. Mattina, T., Palumbo, O., Stallone, R., Pulvirenti, R.M., Di Dio, L., Pavone, P., Carella, M., Pavone, L., 2012. Interstitial 16p13.3 microduplication: case report and critical review of genotype-phenotype correlation. Eur. J. Med. Genet. 55 (12), 747e752. Movahhedian, H.R., Kashani, I.A., Sine, D., Bull, D., Jones, K.L., Rothman, A., 1998. Pulmonary hypertension and trisomy 16. Pediatr. Cardiol. 19 (2), 187e189. Rochat, M.K., Riegel, M., Schinzel, A.A., 2007. Long-term follow-up of a 26-year-old male with duplication of 16p: clinical report and review. Am J Med Genet A. 15 143 (4), 399e408. Stallings, R.L., Doggett, N.A., Okumura, K., Ward, D.C., 1992. Chromosome 16-specific repetitive DNA sequences that map to chromosomal regions known to undergo breakage/rearrangement in leukemia cells. Genomics 13 (2), 332e338. Tassano, E., Alpigiani, M.G., Calcagno, A., Salvati, P., De Miglio, L., Fiorio, P., Cuoco, C., Gimelli, G., 2015. Clinical and molecular delineation of a 16p13.2p13.13 microduplication. Eur. J. Med. Genet. 58 (3), 194e198. na, F., Breckpot, J., Philip, N., Menten, B., Van Esch, H., Scalais, E., Thienpont, B., Be Salamone, J.M., Fong, C.T., Kussmann, J.L., Grange, D.K., Gorski, J.L., Zahir, F., Yong, S.L., Morris, M.M., Gimelli, S., Fryns, J.P., Mortier, G., Friedman, J.M., Villard, L., Bottani, A., Vermeesch, J.R., Cheung, S.W., Devriendt, K., 2010. Duplications of the critical Rubinstein-Taybi deletion region on chromosome 16p13.3 cause a novel recognisable syndrome. J. Med. Genet. 47 (3), 155e161.
Web resources DECIPHER: http://decipher.sanger.ac.uk/.
Please cite this article in press as: Ciaccio, C., et al., 16p13 microduplication without CREBBP involvement: Moving toward a phenotype delineation, European Journal of Medical Genetics (2016), http://dx.doi.org/10.1016/j.ejmg.2016.12.006
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
16p13 microduplication without CREBBP involvement: Moving toward a phenotype delineation Claudia Ciaccio a, *, Arianna Tucci a, Giulietta Scuvera a, Margherita Estienne b, Susanna Esposito a, Donatella Milani a degli Studi di Milano, Fondazione IRCCS Ca' Granda Pediatric Highly Intensive Care Unit, Department of Pathophysiology and Transplantation, Universita Ospedale Maggiore Policlinico, Via Commenda 9, 20122, Milan, Italy b Foundation IRCCS Neurological Institute Carlo Besta, Milan, Italy a
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 June 2016 Received in revised form 12 December 2016 Accepted 16 December 2016 Available online xxx
The short arm of chromosome 16 is one of the less stable regions of our genome, as over 10% of the euchromatic region of 16p is composed of highly complex low copy repeats that are known to be predisposed to rearrangements mediated by non-allelic homologous recombination. The 16p13.3p13.13 molecular region has been defined as the 16p duplication hotspot, and duplications of chromosome 16p13 have recently been confirmed to cause a recognizable syndrome, with CREBBP being the main phenotype-causing gene. To date, only one case report is present in the literature with a 16p13 duplication without CREBBP involvement; we describe here a second analogous case with a not previously reported 16p13.2p13.13 microduplication. This paper allows us to better delineate the clinical features of 16p13 microduplications that do not encompass CREBBP and, concurrently, to narrow the molecular region responsible for congenital heart defects in 16p duplications as well as to propose GRIN2A as a candidate gene for epilepsy. © 2016 Elsevier Masson SAS. All rights reserved.
Keywords: 16p13 microduplication Genotype-phenotype correlation GRIN2A Epilepsy
1. Introduction Partial trisomy 16p is a rare chromosome imbalance that has been well described in the literature. This condition is characterized by global developmental delay (DD), intellectual disability (ID), facial dysmorphisms, and congenital anomalies that can affect any apparatus. The mortality rate in infancy is approximately 50%, but rare long-term survivors have been reported (Martin et al., 2002). The short arm of chromosome 16 is rich in low copy repeats (LCRs), which are known to predispose to rearrangements mediated by non-allelic homologous recombination (NAHR) of the molecular regions they flank; over 10% of the euchromatic region of the short arm of chromosome 16 is in fact composed of highly complex LCRs (Martin et al., 2004). In particular, an unusual and complex class of segmental duplications has been identified whose distribution is, for the most part, specific to the short arm of
* Corresponding author. Pediatric Highly Intensive Care Unit, Department of Pathophysiology and Transplantation, Universit a degli Studi di Milano, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Via della Commenda 9, 20122 Milano, Italy. E-mail address: [email protected] (C. Ciaccio).
chromosome 16 (Stallings et al., 1992). The most prolific chromosome-specific duplication on chromosome 16, termed LCR16a, is a 20 Kb duplication distributed in a non-tandem fashion to multiple locations along the entire length of chromosome 16, including cytogenetic band positions 16q22.2, 16q23, 16p11, 16p12.2, 16p12.3, 16p13.1, and 16p13.3 (Eichler et al., 2011). This duplication may potentially lead to NAHR in more than one region of the short arm of chromosome 16, explaining the high variability of breakpoints and size of the 16p microdeletions and microduplications. The most frequently implicated region in small chromosome rearrangements, defined as the 16p duplication hotspot, is the 16p13.3p13.13 segment (Digilio et al., 2009; Martin et al., 2002). Patients with a duplication of the critical 16p13.3p13.13 molecular region show clinical features that are similar to those shown by patients carrying bigger duplications (Digilio et al., 2009; Tassano et al., 2015). Recurrent clinical findings in the patients affected by this condition are mild to moderate ID, pre- and postnatal growth retardation, hypotonia, and speech and motor delay (language can also be absent). Facial dysmorphisms are distinct and some of them seem to follow an inverted pattern when compared to those shown by individuals with the opposite molecular defect (16p13.3 deletion) (Hammond et al., 2014). Patients
http://dx.doi.org/10.1016/j.ejmg.2016.12.006 1769-7212/© 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Ciaccio, C., et al., 16p13 microduplication without CREBBP involvement: Moving toward a phenotype delineation, European Journal of Medical Genetics (2016), http://dx.doi.org/10.1016/j.ejmg.2016.12.006
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can show microcephaly, ear anomalies, hypertelorism, upslanting and narrow palpebral fissures, prominent glabella, flat nasal bridge, short nose, anteverted nares, flat philtrum, cleft palate, thin lips and micrognathia. The neurological findings include autism spectrum disorder, attention deficit/hyperactivity disorder (ADHD), epilepsy, and brain MRI abnormalities. Any apparatus can potentially be involved: cardiovascular problems (i.e., septal defects, patent ductus arteriosus, mitral valve prolapse, pulmonary insufficiency/stenosis/hypertension), genito-urinary malformations (i.e., renal hypoplasia, vesicourethral reflux, cryptorchidism, small penis, shawl scrotum), skeletal anomalies (i.e., scoliosis, winged scapulae, pectus excavatum, metacarpal bones and phalanx hypoplasia, small hands and feet, camptodactyly, arthrogryposis), refraction defects, and macroglossia have all been reported. This latter aspect, in association with facial muscle hypotonia, can explain the typical tendency to hold the mouth open. Less common findings include inguinal or umbilical hernia, irides dyschromia, dental anomalies (crowding or small teeth), joint hyperlaxity, and different minor skeletal anomalies that are rarely observed (Demeer et al., 2013; Digilio et al., 2009; Li et al., 2013; Marangi et al., 2008; Martin et al., 2002; Mattina et al., 2012; Rochat et al., 2007; Thienpont et al., 2010). All the 16p13.3p13.13 duplications described thus far, except for one, include the CREBBP gene (chromosome 16:3,775,0563,930,121), whose deletion or mutation is responsible for Rubinstein-Taybi Syndrome type 1 (RSTS, OMIM #180849). Duplications of chromosome 16p13.3 encompassing CREBBP have therefore been proposed and later confirmed to cause a recognizable syndrome, with CREBBP being the main gene (Demeer et al., 2013; Marangi et al., 2008; Mattina et al., 2012; Thienpont et al., 2010). The prevalence of this rearrangement has been estimated to account for around 0.043% of all cases of DD (Thienpont et al., 2010). To date, only one patient has been described carrying a 16p13.3p13.13 duplication without CREBBP involvement (Tassano et al., 2015). The patient shares most of the clinical attributes reported in patients carrying duplications in the classical 16p13.3p13.13 hotspot, though the phenotype is milder. Here, we report the case of a girl referred to our Center for a pathological Array-CGH analysis who was found to have a 16p13.2p13.13 microduplication. This report allows us to better delineate the clinical features of 16p13 microduplications not encompassing CREBBP and, concurrently, to narrow the molecular region responsible for congenital heart defects in patients with 16p duplications as well as to propose GRIN2A as a candidate gene for epilepsy.
abnormalities and an absence of epilepsy suggestive waves. An awake EEG performed a year later substantially confirmed the previous result, showing signal asymmetry and occasional slow waves in the anterior regions. Brain MRI (performed at 6 years) was normal. At the age of 7, the girl underwent a neuropsychiatric evaluation, confirming the diagnosis of ID and mood and behavior disorder. A full scale IQ test (e.g. WISC-V) cannot be performed because of the oppositional defiant behavior of the girl. She started anti-psychotic treatment with risperidone, but she never obtained full control of the symptoms. Genetic tests were performed: karyotype, telomere FISH analysis, and FMR1 molecular analysis were all negative. Clinical evaluation at 8 years old revealed growth parameters at the lower centiles of the normal range: height 121 cm (25th centile), weight 19,5 kg (3rd centile), height circumference 39 cm (3e10th centile). She showed a few non-specific facial dysmorphisms, including micrognathia, palpebral ptosis, sparse eyebrows, short philtrum, and thin superior lip (Fig. 1). Mild muscle hypotonia and hyperactivity were present; language was poor and incoherent. Peculiarly, she presented hypotonia and defective control of tongue and mouth muscles, leading to sialorrhea. The Array-CGH analysis was performed in an external laboratory, according to standard protocol, using an oligonucleotide-array with an average resolution of 180 Kb (assembly NCBI Build 36.1-hg 18, March 2006). The analysis showed a 1.63 Mb duplication at 16p13.2p13.13 (arr16p13.2p13.13 (9,598,196-11,231,095)x3) encompassing 6 known OMIM genes: GRIN2A (OMIM*138253), ATF7IP2 (OMIM*613645), EMP2 (OMIM*602334), NUBP1 (OMIM*600280), CIITA (OMIM*600005) and CLEC16A (OMIM*611303) (Fig. 2). qPCR was administered to confirm the cytogenetic finding, the Array-CGH analysis performed in the
2. Clinical report Our patient is an 8-year-old girl who came to our attention because of a pathological result from an Array-CGH analysis, which was performed for psychomotor delay and ID. She was the second child of healthy non-consanguineous parents; the family history was negative for ID and any other genetic conditions. She was born at term and did not experience perinatal distress; the neonatal parameters were within the normal range (length 51 cm, weight 3960 g, head circumference 35 cm). Her growth had always been normal for her height and weight. Mild psychomotor delay was present: she was able to walk independently and uttered her first words at 24 months. Inappropriate behavior had been present since first infancy, with attention deficit, mood disorder and generalized anxiety disorder (GAD). Moreover, she experienced absence epilepsy episodes, with a mean duration of 7e8 min and progressively increasing frequency. An EEG sleep analysis, performed at the age of 4, showed non-specific
Fig. 1. Facial appearance of the patient: low hairline, micrognathia, hypertelorism, mild palpebral ptosis, short philtrum, thin superior lip.
Please cite this article in press as: Ciaccio, C., et al., 16p13 microduplication without CREBBP involvement: Moving toward a phenotype delineation, European Journal of Medical Genetics (2016), http://dx.doi.org/10.1016/j.ejmg.2016.12.006
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Fig. 2. Map of genetic duplication in our patient and in patient previously described by Tassano et al. (2015). The results of the Array-CGH analysis have been uploaded to the UCSC Genome browser (GRCh37 Feb. 2009, hg 19).
parents was negative, confirming that the duplication occurred de novo. 3. Discussion The patient here described is the first case of 16p13.2p13.13 duplication that has ever been reported; it therefore allows us to better delineate the phenotype of 16p13 microduplication not involving CREBBP, condition that has to date been reported in only one patient, by Tassano et al. (2015). There are multiple duplications listed in Decipher with analogous (264947) or smaller dimensions (268461, 263932, 271072, 275724, 314649, and 289949). For most of those duplications, clinical data are not available. In general, they show psychomotor delay/ID, behavioral anomalies, microcephaly, and not better defined facial dysmorphisms. A clinical comparison between our patient and the patient reported by Tassano et al. (2015) is shown in Table 1, while a molecular comparison is shown in Fig. 2. The two patients share most of their features with patients carrying the classic 16p13.13p13.3 microduplication. DD is present in both cases, as well as ID, language delay, and hypotonia. This latter feature may contribute to the attitude of holding the mouth open that is exhibited by our patients. They show common facial dysmorphisms such as sparse eyebrows, palpebral ptosis, a short
philtrum, and a thin superior lip. Additional ocular abnormalities are present in both patients, but they seem to be of a non-specific fashion. Our patient presents hypertelorism (a feature that is also described in patients carrying the classical 16p13.13p13.3 duplication) and a lack of the inferomedial eyelashes (never previously reported); otherwise, the patient reported by Tassano et al. (2015) shows strabismus and right coloboma and macrophthalmia. Cardiovascular anomalies (septal defects, patent ductus arteriosus, mitral valve prolapse, pulmonary insufficiency/stenosis/hypertension) are well represented in patients with the classical 16p13.13p13.3 duplication or with bigger 16p13 duplications (Digilio et al., 2009; Li et al., 2013; Mattina et al., 2012; Rochat et al., 2007; Thienpont et al., 2010) but are absent in both current patients. We can therefore speculate that the region responsible for cardiovascular involvement falls outside the breakpoints of the duplication carried by patient 2. This finding may be of particular interest for the patients' prognosis, as idiopathic pulmonary hypertension appears to be the most severe complication of 16p distal duplications, and it has been previously reported as the primary cause of morbidity in a child carrying a 16p duplication (Movahhedian et al., 1998). The neuropsychiatric profile is similar and is characterized by attention deficit disorder and hyperphagia. In spite of that latter element, our patients' weight was at the 3rd centile, while patient 2 is overweight. Epilepsy is present in both
Table 1 Phenotypic comparison of patients carrying a subtelomeric 16p duplication.
Site (Hg19) and size of 16p duplication
Sex/age at evaluation ID GDD SGA Poor growth Hypotonia Micrognathia Ear anomalies Palpebral ptosis Sparse eyebrows Other ocular features Short philtrum Superior lip anomalies Epilepsy EEG anomalies Brain MRI anomalies Neuropsychiatric profile CV anomalies Skeletal anomalies
Patient 1 Present case
Patient 2 Tassano et al., 2015
arr16p13.2p13.13 (9,598,196-11,231,095)x3 1.63 Mb F/8 y þ þ þ þ þ þ þ Hypertelorism, absence of inferomedial eyelashes þ Thin vermilion Absence epilepsy Non-specific asymmetry Attention deficit, hyperactivity, behavior and mood disorder, GAD, hyperphagia
arr16p13.3p13.13 (6,754,986-12,046383)x3 5.3 Mb F/10.5 þ þ þ þ þ þ þ Right coloboma and macrophthalmia, strabismus þ Thin vermilion Late-onset benign childhood occipital epilepsy Moderate non-specific abnormalities after seizures episodes Ventriculomegaly, corpus callosum agenesis, colpocephaly Attention deficit, hetero-aggression, hyperphagia Butterfly D9-D10-D11
Abbreviations: Pt.: patient; GDD: global developmental delay; SGA: small for gestational age; GAD: generalized anxiety disorder; CV: cardiovascular.
Please cite this article in press as: Ciaccio, C., et al., 16p13 microduplication without CREBBP involvement: Moving toward a phenotype delineation, European Journal of Medical Genetics (2016), http://dx.doi.org/10.1016/j.ejmg.2016.12.006
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patients and is associated with non-specific EEG abnormalities; patient 2 also exhibits brain MRI anomalies (a common feature of the classical 16p13.13p13.3 microduplication). In both cases, the molecular region of the duplication includes the GRIN2A gene, which is not disrupted but is entirely duplicated. GRIN2A is a wellknown and widely studied gene as it encodes the alpha subunit of the ionotropic N-methyl-D-aspartate (NMDA) receptor; this ligandgated receptor is permeable to calcium ions, and its activation results in a calcium influx into post-synaptic cells, with the consequent trigger of several signaling cascades. The NMDA receptor determines key features of the synaptic response and has important consequences for synaptic plasticity and cell physiology (Kalia et al., 2008). In recent years, mutations of the GRIN2A gene have been associated with epilepsy, ID and language impairment (Lesca et al., 2013; Lemke et al., 2013). Lesca et al. (2013) identified a variety of GRIN2A mutations, mostly missense, as one of the major causes of familial epilepsy. Duplications of the gene encoding the alpha subunit of the NDMA receptor could potentially influence the final product at any level, altering the fine modulation of glutamate signaling. Supporting this hypothesis, both patients suffer from epilepsy, as reported for patients carrying GRIN2A mutations. In their paper, Tassano et al. already proposed GRIN2A or ABAT, both of which are within the 16p13 duplicated region of their patient, as possible causal genes for epilepsy. The duplication reported here does not encompass the ABAT gene; we can therefore infer that GRIN2A is the major gene implicated in the epileptic phenotype of these patients. In any case, the lack of functional studies about GRIN2A duplications preclude our going beyond this speculation. Further reports of patients carrying a 16p13 microduplication without CREBBP involvement are necessary to better delineate the phenotype and to confirm our cardiovascular and neurological data. Going forward, a better knowledge of this peculiar chromosomal region will be essential to establishing a patient-tailored follow-up, based also on the molecular defect. Acknowledgments The authors thank the patient and her parents for their cooperation during the diagnostic process. No funding was active on this study. All authors declare that there is no conflict of interest concerning this work. References Demeer, B., Andrieux, J., Receveur, A., Morin, G., Petit, F., Julia, S., Plessis, G., Martin€rs, K., Le Coignard, D., Delobel, B., Firth, H.V., Thuresson, A.C., Lanco Dosen, S., Sjo Caignec, C., Devriendt, K., Mathieu-Dramard, M., 2013. Duplication 16p13.3 and the CREBBP gene: confirmation of the phenotype. Eur. J. Med. Genet. 56 (1), 26e31. Digilio, M.C., Bernardini, L., Capalbo, A., Capolino, R., Gagliardi, M.G., Marino, B., Novelli, A., Dallapiccola, B., 2009. 16p subtelomeric duplication: a clinically recognizable syndrome. Eur. J. Hum. Genet. 17 (9), 1135e1140. Eichler, E.E., Johnson, M.E., Alkan, C., Tuzun, E., Sahinalp, C., Misceo, D., Archidiacono, N., Rocchi, M., 2011. Divergent origins and concerted expansion of two segmental duplications on chromosome 16. J. Hered. 92 (6), 462e468.
Hammond, P., McKee, S., Suttie, M., Allanson, J., Cobben, J.M., Maas, S.M., Quarrell, O., Smith, A.C., Lewis, S., Tassabehji, M., Sisodiya, S., Mattina, T., Hennekam, R., 2014. Opposite effects on facial morphology due to gene dosage sensitivity. Hum. Genet. 133 (9), 1117e1125. Kalia, L.V., Kalia, S.K., Salter, M.W., 2008. NMDA receptors in clinical neurology: excitatory times ahead. Lancet Neurol. 7 (8), 742e755. Lemke, J.R., Lal, D., Reinthaler, E.M., Steiner, I., Nothnagel, M., Alber, M., Geider, K., Laube, B., Schwake, M., Finsterwalder, K., Franke, A., Schilhabel, M., J€ ahn, J.A., Muhle, H., Boor, R., Van Paesschen, W., Caraballo, R., Fejerman, N., Weckhuysen, S., De Jonghe, P., Larsen, J., Møller, R.S., Hjalgrim, H., Addis, L., Tang, S., Hughes, E., Pal, D.K., Veri, K., Vaher, U., Talvik, T., Dimova, P., Guerrero pez, R., Serratosa, J.M., Linnankivi, T., Lehesjoki, A.E., Ruf, S., Wolff, M., Lo Buerki, S., Wohlrab, G., Kroell, J., Datta, A.N., Fiedler, B., Kurlemann, G., Kluger, G., Hahn, A., Haberlandt, D.E., Kutzer, C., Sperner, J., Becker, F., € ck, H., Neophythou, B., Ronen, G.M., GruberWeber, Y.G., Feucht, M., Steinbo Sedlmayr, U., Geldner, J., Harvey, R.J., Hoffmann, P., Herms, S., Altmüller, J., Toliat, M.R., Thiele, H., Nürnberg, P., Wilhelm, C., Stephani, U., Helbig, I., Lerche, H., Zimprich, F., Neubauer, B.A., Biskup, S., von Spiczak, S., 2013. Mutations in GRIN2A cause idiopathic focal epilepsy with rolandic spikes. Nat. Genet. 45, 1067e1072. Lesca, G., Rudolf, G., Bruneau, N., Lozovaya, N., Labalme, A., Boutry-Kryza, N., Salmi, M., Tsintsadze, T., Addis, L., Motte, J., Wright, S., Tsintsadze, V., Michel, A., Doummar, D., Lascelles, K., Strug, L., Waters, P., de Bellescize, J., Vrielynck, P., de Saint Martin, A., Ville, D., Ryvlin, P., Arzimanoglou, A., Hirsch, E., Vincent, A., Pal, D., Burnashev, N., Sanlaville, D., Szepetowski, P., 2013. GRIN2A mutations in acquired epileptic aphasia and related childhood focal epilepsies and encephalopathies with speech and language dysfunction. Nat. Genet. 45 (9), 1061e1066. Li, Z., Liu, J., Li, H., Peng, Y., Lv, W., Long, Z., Liang, D., Wu, L., 2013. Phenotypic expansion of the interstitial 16p13.3 duplication: a case report and review of the literature. Gene 531 (2), 502e505. Marangi, G., Leuzzi, V., Orteschi, D., Grimaldi, M.E., Lecce, R., Neri, G., Zollino, M., 2008. Duplication of the Rubinstein-Taybi region on 16p13.3 is associated with a distinctive phenotype. Am. J. Med. Genet. A 146A (18), 2313e2317. Martin, C.L., Waggoner, D.J., Wong, A., Uhrig, S., Roseberry, J.A., Hedrick, J.F., Pack, S.D., Russell, K., Zackai, E., Dobyns, W.B., Ledbetter, D.H., 2002. “Molecular rulers” for calibrating phenotypic effects of telomere imbalance. J. Med. Genet. 39 (10), 734e740. Martin, J., Han, C., Gordon, L.A., Terry, A., Prabhakar, S., She, X., et al., 2004. The sequence and analysis of duplication-rich human chromosome 16. Nature 432, 988e994. Mattina, T., Palumbo, O., Stallone, R., Pulvirenti, R.M., Di Dio, L., Pavone, P., Carella, M., Pavone, L., 2012. Interstitial 16p13.3 microduplication: case report and critical review of genotype-phenotype correlation. Eur. J. Med. Genet. 55 (12), 747e752. Movahhedian, H.R., Kashani, I.A., Sine, D., Bull, D., Jones, K.L., Rothman, A., 1998. Pulmonary hypertension and trisomy 16. Pediatr. Cardiol. 19 (2), 187e189. Rochat, M.K., Riegel, M., Schinzel, A.A., 2007. Long-term follow-up of a 26-year-old male with duplication of 16p: clinical report and review. Am J Med Genet A. 15 143 (4), 399e408. Stallings, R.L., Doggett, N.A., Okumura, K., Ward, D.C., 1992. Chromosome 16-specific repetitive DNA sequences that map to chromosomal regions known to undergo breakage/rearrangement in leukemia cells. Genomics 13 (2), 332e338. Tassano, E., Alpigiani, M.G., Calcagno, A., Salvati, P., De Miglio, L., Fiorio, P., Cuoco, C., Gimelli, G., 2015. Clinical and molecular delineation of a 16p13.2p13.13 microduplication. Eur. J. Med. Genet. 58 (3), 194e198. na, F., Breckpot, J., Philip, N., Menten, B., Van Esch, H., Scalais, E., Thienpont, B., Be Salamone, J.M., Fong, C.T., Kussmann, J.L., Grange, D.K., Gorski, J.L., Zahir, F., Yong, S.L., Morris, M.M., Gimelli, S., Fryns, J.P., Mortier, G., Friedman, J.M., Villard, L., Bottani, A., Vermeesch, J.R., Cheung, S.W., Devriendt, K., 2010. Duplications of the critical Rubinstein-Taybi deletion region on chromosome 16p13.3 cause a novel recognisable syndrome. J. Med. Genet. 47 (3), 155e161.
Web resources DECIPHER: http://decipher.sanger.ac.uk/.
Please cite this article in press as: Ciaccio, C., et al., 16p13 microduplication without CREBBP involvement: Moving toward a phenotype delineation, European Journal of Medical Genetics (2016), http://dx.doi.org/10.1016/j.ejmg.2016.12.006