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Interstitial deletion of chromosome 2p15-16.1: Report of two patients and critical review of current genotype–phenotype correlation
European Journal of Medical Genetics 55 (2012) 238e244
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European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Original article
Interstitial deletion of chromosome 2p15-16.1: Report of two patients and critical review of current genotypeephenotype correlation Maria Piccione a, *, Ettore Piro a,1, Francesca Serraino a,1, Simona Cavani b, 2, Roberto Ciccone c, 3, Michela Malacarne b, 2, Mauro Pierluigi b, 2, Marianna Vitaloni c, 3, Orsetta Zuffardi c, 3, Giovanni Corsello a, 4 a b c
U.O. Pediatria e TIN Dipartimento Materno-Infantile, Università degli Studi di Palermo via Alfonso Giordano 3, 90127 Palermo, Italy Laboratorio di Genetica, E.O. Galliera, Via A. Volta 6, Genova 16128, Italy Patologia Umana ed Ereditaria. Sez Biologia e Genetica, Università degli Studi di Pavia, Via Forlanini 14 27100, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 April 2011 Accepted 30 January 2012 Available online 18 February 2012
We report two individuals with developmental delay and dysmorphic features, in whom array-based comparative genomic hybridization (array CGH) led to the identification of a 2p15p16.1 de novo deletion. In the first patient (Patient 1) a familial deletion of 6q12, inherited from her father, was also detected. In the second patient (Patient 2) in addition to the 2p15p16.1 microdeletion a de novo deletion in Xq28 was detected. Both individuals shared dysmorphic features and developmental delay with the six reported patients with a 2p15p16.1 microdeletion described in medical literature. Conclusion: in the first patient a 642 kb 2p16.1 deletion (from 60.604 to 61.246 Mb), and a 930 kb 6q12 familial deletion, was detected and in the second a 2.5 Mb 2p15p16.1 deletion (from 60.258 to 62.763 Mb), with a Xq28 deletion, was discovered. The common dysmorphic features and neurodevelopmental delay found in these patients are in agreement with the clinical phenotype of a microdeletion syndrome involving 2p15p16.1. Our data confirm the hypothesis suggesting that 2p15p16.1 deletion is a contiguous gene syndrome. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: Array CGH Developmental delay Dysmorphic features Microdeletion 2p15p16.1 syndrome
1. Introduction Intellectual disability (ID) and behavioral problems can be caused by cryptic chromosome abnormalities including subtelomeric rearrangements. Conventional karyotyping is often unable to identify submicroscopic chromosomal changes. The advent of new technologies, like array-based comparative genomic hybridization (array CGH), has increased the number of microdeletions and microduplications associated with phenotypic features, frequently including idiopathic intellectual disability (ID) [1]. New high resolution genetic analysis techniques often provide an explanation or a diagnosis of abnormal conditions and help us to
find a more precise characterization of genotype/phenotype correlation in childhood chromosomal anomalies. Up to now seven patients with del(2)(p15p16.1) have been identified by array CGH [2e7]. They presented with intellectual disability and dysmorphic facial features such as shortened palpebral fissures, widened inner canthal distance, epicanthal folds, large ears and broad and high nasal root and tip. Autism spectrum disorders have been also described in patients with del(2)(p15p16.1) [8]. Here we describe two individuals with ID and dysmorphic features, in whom the array CGH led to the identification of a de novo deletion of 2p15p16.1. 2. Clinical description
* Corresponding author. Tel.: þ39 916555440; fax: þ39 916555429. E-mail addresses: [email protected] (M. Piccione), [email protected] (E. Piro), [email protected] (F. Serraino), [email protected] (S. Cavani), [email protected] (R. Ciccone), [email protected] (M. Malacarne), [email protected] (M. Pierluigi), [email protected] (M. Vitaloni), [email protected] (O. Zuffardi), [email protected] (G. Corsello). 1 Tel.: þ39 91 6555456. 2 Tel.: þ39 10 5634375. 3 Tel.: þ39 382 987733. 4 Tel.: þ39 91 6555425. 1769-7212/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmg.2012.01.014
2.1. Patient 1 Patient 1 is a 2-year-old female, second child of unrelated healthy Italian parents (mother 38 years old, father 49 years old), born by cesarean section after 38 weeks of gestation. Apgar scores were 7 and 8 at 1 and 5 min respectively. Weight was 4100 g (90th centile), length 51 cm (50th centile) and head circumference 34.5 cm (5th centile). Dysmorphic features consisted of flat facial profile,
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Fig. 1. (a) Clinical features of patient 1. Flat facial profile, thin upper lip (b) Clinical features of patient 2: trigonocephaly with ridging of the metopic suture, severe bitemporal narrowing with elongated face, low set and posteriorly rotated ears, macrostomia.
convergent strabismus, downslanting palpebral fissures, inner canthal distance of 3.5 cm (>97 th centile), epicanthal folds, depressed nasal root, long philtrum, thin upper lip (Fig. 1A). She also showed hypoplasia of the labia minora. Mental development index score on the Bayley II Scale (MDI) was 52, in the range of severely delayed performance with mixed receptive-expressive language delay. Psychomotor development index on the Bayley II Scale (PDI) was 79, in the range of mild delayed performance; neurological examination showed a mild generalized hypotonia of central origin. She developed mild microcephaly (45.8 cm), in contrast with normal length and weight growth velocities. A brain MRI was normal. Cardiologic evaluation including electrocardiogram and echocardiography and abdominal ultrasound investigations were normal. Brainstem Auditory Evoked Response (BAER) and Flash Visual Evoked Potentials (FVEP) were within normal limits. At the age of 4 years Childhood Autism Rating Scale (CARS) [9] did not reveal autistic features. The child behavior profile, assessed by Child Behavior Checklist for Ages 1.5e5 (Achenbach T. 2000 ASEBA University of Vermont) [10] showed the presence of mild externalizing behavior problems with attention deficit and aggressiveness. The child was defined by her parents as defiant, disobedient and uncooperative. 2.2. Patient 2 Patient 2 is a 4-month-old male, first child of unrelated healthy Italian parents (mother 28 years old, father 33 years old), with intrauterine growth restriction (IUGR) and a prenatal suspected diagnosis of duodenal atresia according to the ultrasound findings of a “double bubble” and polyhydramnios (at 35 weeks of gestation). No prenatal testing (maternal serum screening or fetal karyotype) was performed. He was born by cesarean section after 35 weeks of gestation. Apgar scores were 8 and 9 at 1 and 5 min respectively. Weight was 1710 g (<3th centile, 50th centile for 31 weeks gestational age), length 42.5 cm (<3th centile, 50th centile for 33 weeks gestational age), head circumference 30.5 cm (<10th centile, 50th centile for 32 weeks gestational age). At birth his dysmorphic features were trigonocephaly with ridging of the metopic suture, normal inner canthal distance (2.4 cm, 50th centile), epicanthal folds, severe bitemporal narrowing with elonged face, low set and posteriorly rotated ears with abnormal helices, anteverted nostrils, smooth and long philtrum,
macrostomia, thin upper lip and everted lower lip, high narrow palate, bilateral camptodactyly of the III and IV fingers and cryptorchidism (Fig. 1B). Trigonocephaly with ridging of the metopic suture may be responsible for the normal inner canthal. Neurological examination showed axial hypotonia (trunk and limb girdle) of central origin; cranial nerve functions were normal. An abdominal X-ray confirmed the presence of a “double bubble” due to a dilated fluid-filled stomach and proximal duodenum. Therefore, a total parenteral nutrition regimen was started. Cardiologic evaluation with electrocardiogram and echocardiography and abdominal ultrasound findings were unremarkable. A transfontanellar ultrasound investigation showed bilateral grade I intraventricular hemorrhage, bilateral periventricular hyperechogenicity with mild symmetrical ventricular dilatation and the persistence of a cyst of the septum pellucidum. An exploratory laparotomy revealed an annular pancreas with an atretic region in the first portion of the duodenum. A side-to-side duodenostomy was then performed. A three-dimensional bony surface computerized tomography (CT) scan of the head confirmed the clinical evidence of metopic synostosis; brain CT scan showed supra and subtentorial bilateral mild ventricular enlargement, marked and diffuse white matter hypodensity and persistence of a cyst of the septum pellucidum and cavum vergae. BAER recordings were obtained only in response to 110-dB nHL intensity click, revealing a severe bilateral conductive hearing impairment and FVEP were abnormal showing a low voltage and prolonged P200 latency (390 msec). At seven months of corrected age the child showed a severe neurodevelopmental delay and brain MRI showed white matter malacia with cerebral atrophy and hypoplastic corpus callosum. Table 1 Patient 1; probes flanking the breakpoints. Probes
Distal nucleotide Proximal nucleotide Cytogenetic Results position (hg18) position (hg18) band
A_14_P113099 A_14_P127877 A_14_P125332 A_14_P133484
59.866.968 60.457.592 61.100.294 61.127.024
59.867.027 60.457.651 61.100.353 61.127.083
2p16.1 2p16.1 2p16.1 2p16.1
þ/þ /þ /þ þ/þ
A_14_P126643 A_14_P128025 A_14_P129092 A_14_P131450
66.761.677 67.314.301 68.244.706 68.383.776
66.761.736 67.314.360 68.244.765 68.383.835
6q12 6q12 6q12 6q12
þ/þ /þ /þ þ/þ
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Table 2 Patient 2; probes flanking the breakpoints. Probes
Distal nucleotide Proximal nucleotide Cytogenetic Results position (hg18) position (hg18) band
A_16_P00385853 A_16_P35697934 A_16_P15678773 A_14_P115269
60,162,373 60,200,784 62,705,722 62,722,603
60,162,432 60,200,843 62,705,781 62,722,662
2p16.1 2p16.1 2p15 2p15
þ/þ /þ /þ þ/þ
A_16_P21639980 A_16_P41850814 A_16_P21640003 A_16_P03796920
154,302,693 154,317,590 154,322,335 154,388,268
154,302,633 154,317,530 154,322,290 154,388,208
Xq28 Xq28 Xq28 Xq28
þ/0 /0 /0 þ/0
3. Materials and methods 3.1. Chromosomal analysis Conventional karyotyping at a resolution of 550 bands of both probands and their parents was performed on metaphase spreads from cultured lymphocytes according to the standard protocols. 3.2. Molecular karyotyping Genomic DNAs were extracted from peripheral blood lymphocytes using Puregene DNA Isolation Kit [QIAGEN, Hilden, D] for patient 1 and her parents and GenElute Blood Genomic DNA kit [SigmaeAldrich, St. Louis, MO] for patient 2 and his parents according to manufacturers’ instructions. Whole genome array CGH was performed using Human Genome CGH Microarray Kit 44K [Agilent Technologies, Santa Clara, CA] with an average resolution of 100 kb (Build36: March 2006-hg18) for patient 1 and
Table 3 STS results. The alleles show a paternal origin of the chromosome carrying the deletion. STS
Proband
Mother
Father
Parental Origins
D2S357 D2S337 D2S2165 D2S2332
210.71 235.10 113.10 149.58
199.16/210.73 234.86/248.83 113.10/119.37 147.65/149.62
201.37 232.84/246.73 117.24/121.36 153.34
Paternal Paternal Paternal Paternal
Human Genome CGH Microarray kit 244A [Agilent Technologies, Santa Clara, CA] with an average resolution of about 20 kb (Build36: March 2006-hg18) for patient 2 and his parents according to manufacturers’ instructions. Images of the arrays were acquired with Agilent scanner G2505B and analyzed with Feature Extraction software v9.5.1 [Agilent Technologies, Santa Clara, CA]. Graphical overviews of results were obtained with CGH Analytics software v3.4.40 [Agilent Technologies, Santa Clara, CA] (Table 1). 3.3. Fluorescence-in situ-hybridization (FISH) Cytogenetic studies of patient 1 and her parents were performed on metaphases derived from cultures of PHA-stimulated peripheral blood lymphocytes. Single copy DNA probes, RP11440P5 (2p16.1), RP11-416L21 (2p16.1), RP11-475B22 (6q12), cloned in BACs, were obtained from Resources for Human Molecular Cytogenetics [11]. FISH was performed as described by Lichter and Cremer [12]. Slides were counterstained with 40 ,6-diamidino2-phenylindole (DAPI)(200 ng/ml) and analyzed by fluorescence microscope Olympus BX70 equipped with a cooled CCD Video Camera Image Point, Photometrics. Image analysis was carried out with PSI MacProbe software.
Fig. 2. Patient 1, array CGH profile of the chromosome 2 showing the 642 kb deletion at 2p16.1.
M. Piccione et al. / European Journal of Medical Genetics 55 (2012) 238e244
3.4. STS analysis In patient 2 genotyping of polymorphic markers was performed by amplification of the DNAs of the trio using primers labeled with fluorescent probes [ABI 5-Fam, 5-Hex] followed by analysis on an ABI 3100 Genetic Analyzer [Applied Biosystems, Foster City, CA]. Size of alleles and area of the peaks were calculated with Genescan software [GeneScan View 1.1] [BMR-Cribi, Padua]. Polymorphic markers were selected from CEPH-Genethon integrated map [13] and the Genetic map index [14]. Locus information and primer sequences were available from the CEPH genotype database [15] (Table 2). 4. Results 4.1. Patient 1 Genome-wide array analysis of the proband showed a 642 kb deletion at 2p [del(2)(p16.1)], ranging from 60,457 Mb to 61,100 Mb, and a 930 kb deletion at 6q [del(6)(q12)], ranging from 67,314 Mb to 68,244 Mb (Build36: March 2006-hg18) (Fig. 2). FISH analysis on the proband and her parents confirmed the rearrangements and showed that the 2p deletion was de novo while the
241
6q deletion was of paternal origin. The de novo 2p deletion harbored five genes: B-cell CLL/lymphoma 11 (BCL11A), poly(A) polymerase gamma (PAPOLG), REL, PUS10 and proximal end of PEX13; and two non-coding RNA (ncRNA) sequences predicted using sequences from RFAM and MiRBase: U1, SRP_euk_arch (Ensembl Map viewer - Build36: March 2006-hg18). The inherited 6q deletion contains no known genes but three non-coding RNA (ncRNA) sequences predicted using sequences from RFAM and MiRBase: U7, AL590874.6, SNORD65 (Ensembl Map viewer Build36: March 2006-hg18). 4.2. Patient 2 Chromosomal analysis of the child resulted in a normal karyotype (46,XY). Genome-wide array analysis showed a 2.5 Mb de novo deletion at 2p [del(2)(p15p16.1)] of the proband, ranging from 60,111 Mb to 62,616 Mb (Build36: March 2006-hg18). Informative markers mapping into the deleted region showed the paternal origin of the chromosome carrying the rearrangement (Table 3). STRP analysis on a region outside the deletions confirmed the biologic parentage and the de novo nature of this rearrangement (Fig. 3). The 29 kb deletion at Xq [del(X)(q28)] affects two genes: one intragenic loss (4 exons) in BRCC3 and an intronic loss in MTCP.
Fig. 3. Patient 2, array CGH profile of the chromosome 2 showing the 2.5 Mb deletion at 2p15-2p16.1.
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This was a de novo deletion extending from 153,959 Mb to 153,988 Mb (Build36: March 2006-hg18). 5. Discussion Patients with 2p15p16.1 microdeletion syndrome were first characterized in literature in 2007 [2e7] with six individuals reported to date. These patients showed systemic features including moderate to severe intellectual disability, spasticity of the lower extremities, microcephaly with associated brain anomalies, optic nerve hypoplasia, ptosis, shortened palpebral fissures, widened inner canthal distance, epicanthal folds, large ears, broad and high nasal root and tip, everted lower lips, renal anomalies (multicystic kidneys and hydronephrosis) and camptodactyly [2e7] Autism spectrum disorders (ASDs) associated genes OTX1 and XPO1 are located within the 2p15p16.1 region [8] (Table 4). We describe two individuals with intellectual disability and dysmorphic features, in whom the array CGH led to the identification of a 2p15p16.1 deletion. Since the deleted region in 6q12 in patient 1 contains no genes [as seen in Map Viewer at the NCBI web site] and was inherited from a phenotypically normal father, it is believed to have no recognizable phenotypic effects. Our first description of two patients with a 2p15p16.1 deletion at such a young age and the availability of images can contribute to the early detection of phenotypic traits characteristic of the syndrome. The 642 kb deletion interval of 2p16.1 (from 60,457 Mb to 61,100 Mb) includes some known genes with a variety of functions such as B-cell CLL/lymphoma 11 (BCL11A), poly(A) polymerase gamma (PAPOLG), REL, PUS13 and proximal end of PEX13. The oncogene BCL11A is specifically expressed in hematopoietic tissue and it plays an important role in B-cell development; array CGH studies have shown gains in chromosome region 2p as the most common imbalance in classical Hodgkin lymphoma (cHL). The
function of poly(A) polymerase gamma (PAPOLG) gene is not fully understood, but studies have highlighted its role in regulating gene expression via translation and mRNA stability. In patient 2 the microdeletion in 2p15p16.1 was found in addition to a de novo deletion in the Xq28 region, that contains (from 153,959 Mb to 153,988 Mb) MTCP and BRCA1/BRCA2containing complex subunit 3 (BRCC3), whose phenotype contribution although not yet described cannot be completely excluded. Deletion interval of 2p15p16.1 (from 60,111 Mb to 62,616) with a size of 2.5 Mb contains some known genes, which are the same genes reported in patient 1, and other genes which are not deleted in patient 1, like betaGal beta-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2 UDP GlcNA), COMMD, FAM161A, activator of heat shock 90 Kda protein ATPase homolog 2 (AHA1), ubiquitin specific peptidase 34 (USP34), chaperonin containing TCP1, subunit 4 (CCT4) and exportin 1 (XPO1). These deleted genes could explain some different clinical features in patient 2. Mutations in the PEX13 gene can lead to Zellweger syndrome, that is the archetypical peroxisome biogenesis disorder characterized by defective import of proteins into the peroxisome, linked to peroxisomal metabolic dysfunction and widespread tissue pathology [4]. PEX13 inactivation in the mouse caused some of the clinical features of the Zellweger syndrome patients, like intrauterine growth restriction, severe hypotonia, impaired swallowing, neonatal death and cerebral anomalies (disordered lamination in the cerebral cortex) [16]. Our patients do not show clinical features typical of Zellweger phenotype but the loss of function of only one copy of PEX13 gene could be responsible for intellectual disability, even if we have to consider that there are other contiguous known and unknown genes in the deleted region of 2p16.1 [4]. The CCT4 gene in vitro plays a role in the folding of actin, tubulin and others cytoskeletal proteins, but its effect in vivo (camptodactyly and metatarsus
Table 4 Comparison of clinical features of patients with a 2p15p16.1 microdeletion (mapping used hg18). Clinical features
Rajcan-Separovic et al 1
Rajcan-Separovic et al 2
Chabchoub et al. (2008)
deLeeuw et al. (2008)
Liang et al. (2009)
Felix at al (2010)
Prontera et al. (2011)
Present patient 1
Present patient 2
Size of deletion Height centile Weight centile OCF centile Downslanting palpebral fissures Ptosis Short palpebral fissures Strabismus Hypertelorism Epicanthal folds Large ears Broad and high nasal root, prominent tip Smooth and long philtrum Smooth upper vermillion border Everted lower lip High narrow palate Hearing loss Campodactyly Metatarsus abductus, Frequent upper respiratory infections Hydronephrosis Hypogonadism Optic nerve hypoplasia Structural brain abnormalities Neurodevelopmental delay Autistic features
4.5 Mb 75e90th 75e90th <<2th
5.7 Mb <3th 5e10th <<2th
570 kb 97th <<3th 75e90th
3.9 Mb <3th 50th <3th þ
3.2 Mb <3th <3th <3th
3.35 Mb <10th <10th <10th
3.5 Mb <3th <3th 3e10th þ
642 kb 50th 90th 5e10th þ
2.5 Mb <3th <3th <10th
þ þ þ þ þ þ
þ þ þ þ þ
þ þ þ
þ þ þ þ þ þ þ
þ þ þ
þ þ
þ þ þ þ þ
þ þ þ
þ
þ þ
þ þ
þ
þ þ
þ
þ þ
þ
þ
þ þ þ þ þ
þ þ þ þ þ
þ þ
þ þ þ
þ
þ þ
þ þ
þ þ þ þ
þ þ þ
þ þ þ þ
þ
þ þ NA
þ
þ
þ þ
þ þ
þ þ
þ
þþ
þ þ
þ
þ
þþ
þþ
þ: feature present; -: feature absent; NA not assessable/assessed. OCF: occipital-frontal circumference.
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a crucial role in early embryogenesis [20]. The beta-1,3-N-acetylglucosaminyltransferase family has different functions: there is evidence that beta 3Gal-T5 plays a relevant role in gastrointestinal and pancreatic tissues development [21,22]. This could justify the gastrointestinal and pancreatic anomalies described only in patient 2. Our patients support the evidence of a newly recognized microdeletion syndrome involving 2p15p16.1. The difference in the clinical findings in these patients is probably due to the different size of chromosomal regions and genes involved in the deletion (Fig. 4) and to the paternal origin of the de novo microdeletion as demonstrated in three patients (Liang, 2009; Felix, 2010 and our patient 2). Although VRK2 and FANCL genes are not deleted in patient described by Liang et al. (2009) and in our patients, they result to be deleted in patient reported by Prontera et al. (2011). The genes mapping in the more telomeric region (60,2e61 Mb) are candidate for optic nerve hypoplasia [7], but this observation is not supported by our patient 2, who shows the main features of the syndrome except for optic nerve hypoplasia and hydronephrosis. Our study confirms that 2p15p16.1 microdeletion causes a contiguous gene syndrome with specific phenotypic features [8]. References
Fig. 4. Genes deleted in the patients with 2p15-16.1 deletion: A Patient 1: 642 kb, BCLA11, PAPOLG, REL, PUS10, PEX13. B Patient 2: 2.5 Mb, BRCC3, VRK2, BCLA11, PAPOLG, PEX13, PUS10, B3GNT2 UDP GlcNA, COMMD, FAM161A, AHA1, USP34, CCT4, XPO1. C Patient in Chabchoub et al., 2008: 570 kb, KIAA1841, Q96HQ8, AHA1, USP34, XPO1. D Patient in deLeeuw et al., 2008: 3.9 Mb, VRK2, FANCL, BCLA11, PAPOLG, XPO1, PEX13, KIAA1841, AHSA2, USP34. E and F Patients 1and 2in Rajcan-Separovic et al., 2007: 4.5 Mb and 5.7 Mb VRK2, FANCL, BCLA11, PAPOLG, XPO1, PEX13. G Patient in Liang et al., 2009: 3.2 Mb, BCLA11, PAPOLG, XPO1, REL, PEX13, USP34,COMMD. H Patient in Felix et al., 2010: 3.35 Mb, BCLA11, PAPOLG, XPO1, REL, PEX13, USP34,COMMD, CCT4, B3GNT2. I Patient in Prontera et al., 2011: 3.5 Mb, VRK2, FANCL.
abductus) has to be demonstrated [6]. XPO1 (Exportin 1) encodes for a protein localized at the level of the nucleoplasm interacting with the nuclear membrane. This protein is essential for proliferation and chromosome maintenance. It has been demonstrated that this protein, also called CMR1 (chromosome region maintenance 1), is involved in the transport of both mRNA and nuclear export signals (NESs) containing proteins [17,18].This protein is highly conserved, it has 52% sequence identity with its Saccharomyces cerevisiae ortholog and 96.7% sequence identity with Xenopus laevis protein. XPO1 appears to be one of the best conserved proteins among vertebrates and this characteristic firmly indicates its crucial role in the regulation of different pathways during development. The essential role of this protein in coordinating nuclear events such as mitosis and transcriptional activation with nuclear transport has been widely demonstrated but now various evidences show that it also plays a crucial role in early embryogenesis. Callanan et al. [19] showed that the X. laevis otholog of the human XPO1 is maternally expressed and is present throughout early development; its activity is tightly regulated as a function of development stage. XPO1, which is initially in an inactive status, becomes functional during the gastrulaeneurula transition (GNT) period. For this reason, the activation of XPO1-signaling must be considered an essential feature of this critical stage of development. In this work they underlined how XPO1 over-expression and inhibition by leptomycin B treatment during neurula stage affect the normal development of embryos. This protein is essential for proliferation and chromosome maintenance and it also plays
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mammalian CRM1, a protein involved in nuclear export of proteins, J. Biol. Chem. 272 (42) (1997) 29742e29751. [19] M. Callanan, N. Kudo, S. Gout, M. Brocard, M. Yoshida, S. Dimitrov, S. Khochbin, Developmentally regulated activity of CRM1/XPO1 during early Xenopus embryogenesis, J. Cell Sci. 113 (2000) 451e459. [20] K. Stade, C.S. Ford, C. Guthrie, K. Weis, Exportin 1 (Crm1p) is an essential nuclear export factor, Cell 90 (6) (1997) 1041e1050.
[21] N. Hayashi, S. Nakamori, J. Okami, H. Nagano, K. Dono, K. Umeshita, M. Sakon, H. Narimatsu, M. Monden, Association between expression levels of CA 19-9 and N-acetylglucosamine-beta;1,3 galactosyltransferase 5 gene in human pancreatic cancer tissue, Pathobiology 71 (1) (2004) 26e34. [22] L. Mare, M. Trinchera, Suppression of beta 1,3 galactosyltransferase beta 3Gal-T5 in cancer cells reduces sialyl-Lewis a and enhances poly N-acetyllactosamines and sialyl-Lewis x on O-glycans, Eur. J. Biochem. 271 (1) (2004) 186e194.
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European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Original article
Interstitial deletion of chromosome 2p15-16.1: Report of two patients and critical review of current genotypeephenotype correlation Maria Piccione a, *, Ettore Piro a,1, Francesca Serraino a,1, Simona Cavani b, 2, Roberto Ciccone c, 3, Michela Malacarne b, 2, Mauro Pierluigi b, 2, Marianna Vitaloni c, 3, Orsetta Zuffardi c, 3, Giovanni Corsello a, 4 a b c
U.O. Pediatria e TIN Dipartimento Materno-Infantile, Università degli Studi di Palermo via Alfonso Giordano 3, 90127 Palermo, Italy Laboratorio di Genetica, E.O. Galliera, Via A. Volta 6, Genova 16128, Italy Patologia Umana ed Ereditaria. Sez Biologia e Genetica, Università degli Studi di Pavia, Via Forlanini 14 27100, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 April 2011 Accepted 30 January 2012 Available online 18 February 2012
We report two individuals with developmental delay and dysmorphic features, in whom array-based comparative genomic hybridization (array CGH) led to the identification of a 2p15p16.1 de novo deletion. In the first patient (Patient 1) a familial deletion of 6q12, inherited from her father, was also detected. In the second patient (Patient 2) in addition to the 2p15p16.1 microdeletion a de novo deletion in Xq28 was detected. Both individuals shared dysmorphic features and developmental delay with the six reported patients with a 2p15p16.1 microdeletion described in medical literature. Conclusion: in the first patient a 642 kb 2p16.1 deletion (from 60.604 to 61.246 Mb), and a 930 kb 6q12 familial deletion, was detected and in the second a 2.5 Mb 2p15p16.1 deletion (from 60.258 to 62.763 Mb), with a Xq28 deletion, was discovered. The common dysmorphic features and neurodevelopmental delay found in these patients are in agreement with the clinical phenotype of a microdeletion syndrome involving 2p15p16.1. Our data confirm the hypothesis suggesting that 2p15p16.1 deletion is a contiguous gene syndrome. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: Array CGH Developmental delay Dysmorphic features Microdeletion 2p15p16.1 syndrome
1. Introduction Intellectual disability (ID) and behavioral problems can be caused by cryptic chromosome abnormalities including subtelomeric rearrangements. Conventional karyotyping is often unable to identify submicroscopic chromosomal changes. The advent of new technologies, like array-based comparative genomic hybridization (array CGH), has increased the number of microdeletions and microduplications associated with phenotypic features, frequently including idiopathic intellectual disability (ID) [1]. New high resolution genetic analysis techniques often provide an explanation or a diagnosis of abnormal conditions and help us to
find a more precise characterization of genotype/phenotype correlation in childhood chromosomal anomalies. Up to now seven patients with del(2)(p15p16.1) have been identified by array CGH [2e7]. They presented with intellectual disability and dysmorphic facial features such as shortened palpebral fissures, widened inner canthal distance, epicanthal folds, large ears and broad and high nasal root and tip. Autism spectrum disorders have been also described in patients with del(2)(p15p16.1) [8]. Here we describe two individuals with ID and dysmorphic features, in whom the array CGH led to the identification of a de novo deletion of 2p15p16.1. 2. Clinical description
* Corresponding author. Tel.: þ39 916555440; fax: þ39 916555429. E-mail addresses: [email protected] (M. Piccione), [email protected] (E. Piro), [email protected] (F. Serraino), [email protected] (S. Cavani), [email protected] (R. Ciccone), [email protected] (M. Malacarne), [email protected] (M. Pierluigi), [email protected] (M. Vitaloni), [email protected] (O. Zuffardi), [email protected] (G. Corsello). 1 Tel.: þ39 91 6555456. 2 Tel.: þ39 10 5634375. 3 Tel.: þ39 382 987733. 4 Tel.: þ39 91 6555425. 1769-7212/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmg.2012.01.014
2.1. Patient 1 Patient 1 is a 2-year-old female, second child of unrelated healthy Italian parents (mother 38 years old, father 49 years old), born by cesarean section after 38 weeks of gestation. Apgar scores were 7 and 8 at 1 and 5 min respectively. Weight was 4100 g (90th centile), length 51 cm (50th centile) and head circumference 34.5 cm (5th centile). Dysmorphic features consisted of flat facial profile,
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Fig. 1. (a) Clinical features of patient 1. Flat facial profile, thin upper lip (b) Clinical features of patient 2: trigonocephaly with ridging of the metopic suture, severe bitemporal narrowing with elongated face, low set and posteriorly rotated ears, macrostomia.
convergent strabismus, downslanting palpebral fissures, inner canthal distance of 3.5 cm (>97 th centile), epicanthal folds, depressed nasal root, long philtrum, thin upper lip (Fig. 1A). She also showed hypoplasia of the labia minora. Mental development index score on the Bayley II Scale (MDI) was 52, in the range of severely delayed performance with mixed receptive-expressive language delay. Psychomotor development index on the Bayley II Scale (PDI) was 79, in the range of mild delayed performance; neurological examination showed a mild generalized hypotonia of central origin. She developed mild microcephaly (45.8 cm), in contrast with normal length and weight growth velocities. A brain MRI was normal. Cardiologic evaluation including electrocardiogram and echocardiography and abdominal ultrasound investigations were normal. Brainstem Auditory Evoked Response (BAER) and Flash Visual Evoked Potentials (FVEP) were within normal limits. At the age of 4 years Childhood Autism Rating Scale (CARS) [9] did not reveal autistic features. The child behavior profile, assessed by Child Behavior Checklist for Ages 1.5e5 (Achenbach T. 2000 ASEBA University of Vermont) [10] showed the presence of mild externalizing behavior problems with attention deficit and aggressiveness. The child was defined by her parents as defiant, disobedient and uncooperative. 2.2. Patient 2 Patient 2 is a 4-month-old male, first child of unrelated healthy Italian parents (mother 28 years old, father 33 years old), with intrauterine growth restriction (IUGR) and a prenatal suspected diagnosis of duodenal atresia according to the ultrasound findings of a “double bubble” and polyhydramnios (at 35 weeks of gestation). No prenatal testing (maternal serum screening or fetal karyotype) was performed. He was born by cesarean section after 35 weeks of gestation. Apgar scores were 8 and 9 at 1 and 5 min respectively. Weight was 1710 g (<3th centile, 50th centile for 31 weeks gestational age), length 42.5 cm (<3th centile, 50th centile for 33 weeks gestational age), head circumference 30.5 cm (<10th centile, 50th centile for 32 weeks gestational age). At birth his dysmorphic features were trigonocephaly with ridging of the metopic suture, normal inner canthal distance (2.4 cm, 50th centile), epicanthal folds, severe bitemporal narrowing with elonged face, low set and posteriorly rotated ears with abnormal helices, anteverted nostrils, smooth and long philtrum,
macrostomia, thin upper lip and everted lower lip, high narrow palate, bilateral camptodactyly of the III and IV fingers and cryptorchidism (Fig. 1B). Trigonocephaly with ridging of the metopic suture may be responsible for the normal inner canthal. Neurological examination showed axial hypotonia (trunk and limb girdle) of central origin; cranial nerve functions were normal. An abdominal X-ray confirmed the presence of a “double bubble” due to a dilated fluid-filled stomach and proximal duodenum. Therefore, a total parenteral nutrition regimen was started. Cardiologic evaluation with electrocardiogram and echocardiography and abdominal ultrasound findings were unremarkable. A transfontanellar ultrasound investigation showed bilateral grade I intraventricular hemorrhage, bilateral periventricular hyperechogenicity with mild symmetrical ventricular dilatation and the persistence of a cyst of the septum pellucidum. An exploratory laparotomy revealed an annular pancreas with an atretic region in the first portion of the duodenum. A side-to-side duodenostomy was then performed. A three-dimensional bony surface computerized tomography (CT) scan of the head confirmed the clinical evidence of metopic synostosis; brain CT scan showed supra and subtentorial bilateral mild ventricular enlargement, marked and diffuse white matter hypodensity and persistence of a cyst of the septum pellucidum and cavum vergae. BAER recordings were obtained only in response to 110-dB nHL intensity click, revealing a severe bilateral conductive hearing impairment and FVEP were abnormal showing a low voltage and prolonged P200 latency (390 msec). At seven months of corrected age the child showed a severe neurodevelopmental delay and brain MRI showed white matter malacia with cerebral atrophy and hypoplastic corpus callosum. Table 1 Patient 1; probes flanking the breakpoints. Probes
Distal nucleotide Proximal nucleotide Cytogenetic Results position (hg18) position (hg18) band
A_14_P113099 A_14_P127877 A_14_P125332 A_14_P133484
59.866.968 60.457.592 61.100.294 61.127.024
59.867.027 60.457.651 61.100.353 61.127.083
2p16.1 2p16.1 2p16.1 2p16.1
þ/þ /þ /þ þ/þ
A_14_P126643 A_14_P128025 A_14_P129092 A_14_P131450
66.761.677 67.314.301 68.244.706 68.383.776
66.761.736 67.314.360 68.244.765 68.383.835
6q12 6q12 6q12 6q12
þ/þ /þ /þ þ/þ
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Table 2 Patient 2; probes flanking the breakpoints. Probes
Distal nucleotide Proximal nucleotide Cytogenetic Results position (hg18) position (hg18) band
A_16_P00385853 A_16_P35697934 A_16_P15678773 A_14_P115269
60,162,373 60,200,784 62,705,722 62,722,603
60,162,432 60,200,843 62,705,781 62,722,662
2p16.1 2p16.1 2p15 2p15
þ/þ /þ /þ þ/þ
A_16_P21639980 A_16_P41850814 A_16_P21640003 A_16_P03796920
154,302,693 154,317,590 154,322,335 154,388,268
154,302,633 154,317,530 154,322,290 154,388,208
Xq28 Xq28 Xq28 Xq28
þ/0 /0 /0 þ/0
3. Materials and methods 3.1. Chromosomal analysis Conventional karyotyping at a resolution of 550 bands of both probands and their parents was performed on metaphase spreads from cultured lymphocytes according to the standard protocols. 3.2. Molecular karyotyping Genomic DNAs were extracted from peripheral blood lymphocytes using Puregene DNA Isolation Kit [QIAGEN, Hilden, D] for patient 1 and her parents and GenElute Blood Genomic DNA kit [SigmaeAldrich, St. Louis, MO] for patient 2 and his parents according to manufacturers’ instructions. Whole genome array CGH was performed using Human Genome CGH Microarray Kit 44K [Agilent Technologies, Santa Clara, CA] with an average resolution of 100 kb (Build36: March 2006-hg18) for patient 1 and
Table 3 STS results. The alleles show a paternal origin of the chromosome carrying the deletion. STS
Proband
Mother
Father
Parental Origins
D2S357 D2S337 D2S2165 D2S2332
210.71 235.10 113.10 149.58
199.16/210.73 234.86/248.83 113.10/119.37 147.65/149.62
201.37 232.84/246.73 117.24/121.36 153.34
Paternal Paternal Paternal Paternal
Human Genome CGH Microarray kit 244A [Agilent Technologies, Santa Clara, CA] with an average resolution of about 20 kb (Build36: March 2006-hg18) for patient 2 and his parents according to manufacturers’ instructions. Images of the arrays were acquired with Agilent scanner G2505B and analyzed with Feature Extraction software v9.5.1 [Agilent Technologies, Santa Clara, CA]. Graphical overviews of results were obtained with CGH Analytics software v3.4.40 [Agilent Technologies, Santa Clara, CA] (Table 1). 3.3. Fluorescence-in situ-hybridization (FISH) Cytogenetic studies of patient 1 and her parents were performed on metaphases derived from cultures of PHA-stimulated peripheral blood lymphocytes. Single copy DNA probes, RP11440P5 (2p16.1), RP11-416L21 (2p16.1), RP11-475B22 (6q12), cloned in BACs, were obtained from Resources for Human Molecular Cytogenetics [11]. FISH was performed as described by Lichter and Cremer [12]. Slides were counterstained with 40 ,6-diamidino2-phenylindole (DAPI)(200 ng/ml) and analyzed by fluorescence microscope Olympus BX70 equipped with a cooled CCD Video Camera Image Point, Photometrics. Image analysis was carried out with PSI MacProbe software.
Fig. 2. Patient 1, array CGH profile of the chromosome 2 showing the 642 kb deletion at 2p16.1.
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3.4. STS analysis In patient 2 genotyping of polymorphic markers was performed by amplification of the DNAs of the trio using primers labeled with fluorescent probes [ABI 5-Fam, 5-Hex] followed by analysis on an ABI 3100 Genetic Analyzer [Applied Biosystems, Foster City, CA]. Size of alleles and area of the peaks were calculated with Genescan software [GeneScan View 1.1] [BMR-Cribi, Padua]. Polymorphic markers were selected from CEPH-Genethon integrated map [13] and the Genetic map index [14]. Locus information and primer sequences were available from the CEPH genotype database [15] (Table 2). 4. Results 4.1. Patient 1 Genome-wide array analysis of the proband showed a 642 kb deletion at 2p [del(2)(p16.1)], ranging from 60,457 Mb to 61,100 Mb, and a 930 kb deletion at 6q [del(6)(q12)], ranging from 67,314 Mb to 68,244 Mb (Build36: March 2006-hg18) (Fig. 2). FISH analysis on the proband and her parents confirmed the rearrangements and showed that the 2p deletion was de novo while the
241
6q deletion was of paternal origin. The de novo 2p deletion harbored five genes: B-cell CLL/lymphoma 11 (BCL11A), poly(A) polymerase gamma (PAPOLG), REL, PUS10 and proximal end of PEX13; and two non-coding RNA (ncRNA) sequences predicted using sequences from RFAM and MiRBase: U1, SRP_euk_arch (Ensembl Map viewer - Build36: March 2006-hg18). The inherited 6q deletion contains no known genes but three non-coding RNA (ncRNA) sequences predicted using sequences from RFAM and MiRBase: U7, AL590874.6, SNORD65 (Ensembl Map viewer Build36: March 2006-hg18). 4.2. Patient 2 Chromosomal analysis of the child resulted in a normal karyotype (46,XY). Genome-wide array analysis showed a 2.5 Mb de novo deletion at 2p [del(2)(p15p16.1)] of the proband, ranging from 60,111 Mb to 62,616 Mb (Build36: March 2006-hg18). Informative markers mapping into the deleted region showed the paternal origin of the chromosome carrying the rearrangement (Table 3). STRP analysis on a region outside the deletions confirmed the biologic parentage and the de novo nature of this rearrangement (Fig. 3). The 29 kb deletion at Xq [del(X)(q28)] affects two genes: one intragenic loss (4 exons) in BRCC3 and an intronic loss in MTCP.
Fig. 3. Patient 2, array CGH profile of the chromosome 2 showing the 2.5 Mb deletion at 2p15-2p16.1.
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This was a de novo deletion extending from 153,959 Mb to 153,988 Mb (Build36: March 2006-hg18). 5. Discussion Patients with 2p15p16.1 microdeletion syndrome were first characterized in literature in 2007 [2e7] with six individuals reported to date. These patients showed systemic features including moderate to severe intellectual disability, spasticity of the lower extremities, microcephaly with associated brain anomalies, optic nerve hypoplasia, ptosis, shortened palpebral fissures, widened inner canthal distance, epicanthal folds, large ears, broad and high nasal root and tip, everted lower lips, renal anomalies (multicystic kidneys and hydronephrosis) and camptodactyly [2e7] Autism spectrum disorders (ASDs) associated genes OTX1 and XPO1 are located within the 2p15p16.1 region [8] (Table 4). We describe two individuals with intellectual disability and dysmorphic features, in whom the array CGH led to the identification of a 2p15p16.1 deletion. Since the deleted region in 6q12 in patient 1 contains no genes [as seen in Map Viewer at the NCBI web site] and was inherited from a phenotypically normal father, it is believed to have no recognizable phenotypic effects. Our first description of two patients with a 2p15p16.1 deletion at such a young age and the availability of images can contribute to the early detection of phenotypic traits characteristic of the syndrome. The 642 kb deletion interval of 2p16.1 (from 60,457 Mb to 61,100 Mb) includes some known genes with a variety of functions such as B-cell CLL/lymphoma 11 (BCL11A), poly(A) polymerase gamma (PAPOLG), REL, PUS13 and proximal end of PEX13. The oncogene BCL11A is specifically expressed in hematopoietic tissue and it plays an important role in B-cell development; array CGH studies have shown gains in chromosome region 2p as the most common imbalance in classical Hodgkin lymphoma (cHL). The
function of poly(A) polymerase gamma (PAPOLG) gene is not fully understood, but studies have highlighted its role in regulating gene expression via translation and mRNA stability. In patient 2 the microdeletion in 2p15p16.1 was found in addition to a de novo deletion in the Xq28 region, that contains (from 153,959 Mb to 153,988 Mb) MTCP and BRCA1/BRCA2containing complex subunit 3 (BRCC3), whose phenotype contribution although not yet described cannot be completely excluded. Deletion interval of 2p15p16.1 (from 60,111 Mb to 62,616) with a size of 2.5 Mb contains some known genes, which are the same genes reported in patient 1, and other genes which are not deleted in patient 1, like betaGal beta-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2 UDP GlcNA), COMMD, FAM161A, activator of heat shock 90 Kda protein ATPase homolog 2 (AHA1), ubiquitin specific peptidase 34 (USP34), chaperonin containing TCP1, subunit 4 (CCT4) and exportin 1 (XPO1). These deleted genes could explain some different clinical features in patient 2. Mutations in the PEX13 gene can lead to Zellweger syndrome, that is the archetypical peroxisome biogenesis disorder characterized by defective import of proteins into the peroxisome, linked to peroxisomal metabolic dysfunction and widespread tissue pathology [4]. PEX13 inactivation in the mouse caused some of the clinical features of the Zellweger syndrome patients, like intrauterine growth restriction, severe hypotonia, impaired swallowing, neonatal death and cerebral anomalies (disordered lamination in the cerebral cortex) [16]. Our patients do not show clinical features typical of Zellweger phenotype but the loss of function of only one copy of PEX13 gene could be responsible for intellectual disability, even if we have to consider that there are other contiguous known and unknown genes in the deleted region of 2p16.1 [4]. The CCT4 gene in vitro plays a role in the folding of actin, tubulin and others cytoskeletal proteins, but its effect in vivo (camptodactyly and metatarsus
Table 4 Comparison of clinical features of patients with a 2p15p16.1 microdeletion (mapping used hg18). Clinical features
Rajcan-Separovic et al 1
Rajcan-Separovic et al 2
Chabchoub et al. (2008)
deLeeuw et al. (2008)
Liang et al. (2009)
Felix at al (2010)
Prontera et al. (2011)
Present patient 1
Present patient 2
Size of deletion Height centile Weight centile OCF centile Downslanting palpebral fissures Ptosis Short palpebral fissures Strabismus Hypertelorism Epicanthal folds Large ears Broad and high nasal root, prominent tip Smooth and long philtrum Smooth upper vermillion border Everted lower lip High narrow palate Hearing loss Campodactyly Metatarsus abductus, Frequent upper respiratory infections Hydronephrosis Hypogonadism Optic nerve hypoplasia Structural brain abnormalities Neurodevelopmental delay Autistic features
4.5 Mb 75e90th 75e90th <<2th
5.7 Mb <3th 5e10th <<2th
570 kb 97th <<3th 75e90th
3.9 Mb <3th 50th <3th þ
3.2 Mb <3th <3th <3th
3.35 Mb <10th <10th <10th
3.5 Mb <3th <3th 3e10th þ
642 kb 50th 90th 5e10th þ
2.5 Mb <3th <3th <10th
þ þ þ þ þ þ
þ þ þ þ þ
þ þ þ
þ þ þ þ þ þ þ
þ þ þ
þ þ
þ þ þ þ þ
þ þ þ
þ
þ þ
þ þ
þ
þ þ
þ
þ þ
þ
þ
þ þ þ þ þ
þ þ þ þ þ
þ þ
þ þ þ
þ
þ þ
þ þ
þ þ þ þ
þ þ þ
þ þ þ þ
þ
þ þ NA
þ
þ
þ þ
þ þ
þ þ
þ
þþ
þ þ
þ
þ
þþ
þþ
þ: feature present; -: feature absent; NA not assessable/assessed. OCF: occipital-frontal circumference.
M. Piccione et al. / European Journal of Medical Genetics 55 (2012) 238e244
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a crucial role in early embryogenesis [20]. The beta-1,3-N-acetylglucosaminyltransferase family has different functions: there is evidence that beta 3Gal-T5 plays a relevant role in gastrointestinal and pancreatic tissues development [21,22]. This could justify the gastrointestinal and pancreatic anomalies described only in patient 2. Our patients support the evidence of a newly recognized microdeletion syndrome involving 2p15p16.1. The difference in the clinical findings in these patients is probably due to the different size of chromosomal regions and genes involved in the deletion (Fig. 4) and to the paternal origin of the de novo microdeletion as demonstrated in three patients (Liang, 2009; Felix, 2010 and our patient 2). Although VRK2 and FANCL genes are not deleted in patient described by Liang et al. (2009) and in our patients, they result to be deleted in patient reported by Prontera et al. (2011). The genes mapping in the more telomeric region (60,2e61 Mb) are candidate for optic nerve hypoplasia [7], but this observation is not supported by our patient 2, who shows the main features of the syndrome except for optic nerve hypoplasia and hydronephrosis. Our study confirms that 2p15p16.1 microdeletion causes a contiguous gene syndrome with specific phenotypic features [8]. References
Fig. 4. Genes deleted in the patients with 2p15-16.1 deletion: A Patient 1: 642 kb, BCLA11, PAPOLG, REL, PUS10, PEX13. B Patient 2: 2.5 Mb, BRCC3, VRK2, BCLA11, PAPOLG, PEX13, PUS10, B3GNT2 UDP GlcNA, COMMD, FAM161A, AHA1, USP34, CCT4, XPO1. C Patient in Chabchoub et al., 2008: 570 kb, KIAA1841, Q96HQ8, AHA1, USP34, XPO1. D Patient in deLeeuw et al., 2008: 3.9 Mb, VRK2, FANCL, BCLA11, PAPOLG, XPO1, PEX13, KIAA1841, AHSA2, USP34. E and F Patients 1and 2in Rajcan-Separovic et al., 2007: 4.5 Mb and 5.7 Mb VRK2, FANCL, BCLA11, PAPOLG, XPO1, PEX13. G Patient in Liang et al., 2009: 3.2 Mb, BCLA11, PAPOLG, XPO1, REL, PEX13, USP34,COMMD. H Patient in Felix et al., 2010: 3.35 Mb, BCLA11, PAPOLG, XPO1, REL, PEX13, USP34,COMMD, CCT4, B3GNT2. I Patient in Prontera et al., 2011: 3.5 Mb, VRK2, FANCL.
abductus) has to be demonstrated [6]. XPO1 (Exportin 1) encodes for a protein localized at the level of the nucleoplasm interacting with the nuclear membrane. This protein is essential for proliferation and chromosome maintenance. It has been demonstrated that this protein, also called CMR1 (chromosome region maintenance 1), is involved in the transport of both mRNA and nuclear export signals (NESs) containing proteins [17,18].This protein is highly conserved, it has 52% sequence identity with its Saccharomyces cerevisiae ortholog and 96.7% sequence identity with Xenopus laevis protein. XPO1 appears to be one of the best conserved proteins among vertebrates and this characteristic firmly indicates its crucial role in the regulation of different pathways during development. The essential role of this protein in coordinating nuclear events such as mitosis and transcriptional activation with nuclear transport has been widely demonstrated but now various evidences show that it also plays a crucial role in early embryogenesis. Callanan et al. [19] showed that the X. laevis otholog of the human XPO1 is maternally expressed and is present throughout early development; its activity is tightly regulated as a function of development stage. XPO1, which is initially in an inactive status, becomes functional during the gastrulaeneurula transition (GNT) period. For this reason, the activation of XPO1-signaling must be considered an essential feature of this critical stage of development. In this work they underlined how XPO1 over-expression and inhibition by leptomycin B treatment during neurula stage affect the normal development of embryos. This protein is essential for proliferation and chromosome maintenance and it also plays
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