Accepted Manuscript Haploinsufficiency of XP01 and USP34 by a de novo 230 kb deletion in 2p15, in a patient with mild intellectual disability and cranio-facial dysmorphisms Madeleine Fannemel , Tuva Barøy , Asbjørn Holmgren , Olaug K. Rødningen , Trine M. Haugsand , Børre Hansen , Eirik Frengen , Doriana Misceo , PhD PII:
S1769-7212(14)00131-1
DOI:
10.1016/j.ejmg.2014.05.008
Reference:
EJMG 2948
To appear in:
European Journal of Medical Genetics
Received Date: 13 January 2014 Accepted Date: 28 May 2014
Please cite this article as: M. Fannemel, T. Barøy, A. Holmgren, O.K. Rødningen, T.M. Haugsand, B. Hansen, E. Frengen, D. Misceo, Haploinsufficiency of XP01 and USP34 by a de novo 230 kb deletion in 2p15, in a patient with mild intellectual disability and cranio-facial dysmorphisms, European Journal of Medical Genetics (2014), doi: 10.1016/j.ejmg.2014.05.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Haploinsufficiency of XP01 and USP34 by a de novo 230 kb deletion in 2p15, in a patient with mild intellectual disability and cranio-facial dysmorphisms Running title: A de novo 230 kb deletion in 2p15 Madeleine Fannemel1, Tuva Barøy1, Asbjørn Holmgren1, Olaug K. Rødningen1, Trine M. Haugsand2,
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Børre Hansen2, Eirik Frengen1, Doriana Misceo1§
Department of Medical Genetics, University of Oslo and Oslo University Hospital, Oslo, Norway
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Department for adult habilitation, Akershus University Hospital, Oslo, Norway
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MF: [email protected] TB: [email protected]
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E-mail addresses:
AH: [email protected]
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OKR: [email protected]
TMH: [email protected]
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BH: [email protected]
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EF: [email protected] DM: [email protected]
§ To whom correspondence should be addressed: Doriana Misceo, PhD, Department of Medical Genetics, University of Oslo, P.O. Box 1036, Blindern, N-0315 Oslo, Norway. [email protected]; telephone: +47 22116228, fax: +47 22119899 Disclosure statement No competing financial interests exist.
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ACCEPTED MANUSCRIPT Abstract 2p15p16.1-deletion syndrome was first described in 2007 based on the clinical presentation of two patients. The syndrome is characterized by intellectual disability, autism spectrum disorders, microcephaly, dysmorphic facial features and a variety of congenital organ defects. The precise
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genotype-phenotype correlation in 2p15- deletion syndrome is not understood. However, greater
insight can be obtained by thorough clinical investigation of patients carrying deletions, especially those of small size. We report a 21-year-old male patient with features overlapping the clinical
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spectrum of the 2p15p16.1 deletion syndrome, such as intellectual disability, dysmorphic facial
features, and congenital defects. He carried a 230 kb de novo deletion (chr2:61500346-61733075 bp,
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hg19), which affects the genes USP34, SNORA70B and XPO1. While there is a lack of functional data on SNORA70B, the involvement of USP34 and XPO1 in the regulation of fundamental developmental processes is well known. We suggest that haploinsuffiency of one or both of these genes is likely to be responsible for the disease in our patient.
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Keywords: congenital anomalies, dysmorphic features, intellectual disability, USP34, XPO1 or CRM1, 2p15p16.1 deletion syndrome. 1. Clinical description
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The patient is a 21-year-old male, single child of a non-consanguineous Norwegian couple. His father was diagnosed with bipolar disorder. Early pregnancy was complicated by small bleedings lasting
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about one week and maternal hyperthyreosis. No drug or alcohol exposure during the pregnancy was reported by the mother. The patient was born at term with a weight of 3240 g (10th-25th centile), a length of 52 cm (75th centile), while occipitofrontal circumference (OFC) was not recorded (Norwegian growth standards are in use [1]). Apgar score was normal. Amniotic fluid was discolored. He started to walk at the age of 13-14 months and spoke his first word at 1 year. There were concerns about his language development from the age of 2. At the age of 4 he scored more than 3 standard deviations (SD) below the age mean in all fields on McCarthy’s scales of children’s ability (MCSA). He was repeatedly evaluated during childhood and he followed a special educational program. An
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ACCEPTED MANUSCRIPT extensive panel of cognitive and behavioral tests were performed at the age of 20 years. The results were consistent with mild intellectual disability (ID). Interestingly, the patient scored 63 on the Wechsler adult intelligence scale (WAIS IV), corresponding to a developmental age of 10-12 years (using a 95% confidence interval), but according to the Adaptive Behavior Assessment System
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(ABAS) he scored in the range of 7-8 years, which is more than 2 SD below the WAIS IV results. At 20 years of age he sought a psychologist due to anxiety, obsessive compulsive actions and ideas. He received no medication for this. At his most recent examination at the age of 21 his height was 174 cm (10th-25th centile), weight 52 kg (10th centile), and OFC 55.2 cm (10th centile). Facial dysmorphic
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features included a high, narrow and sloping forehead, possibly due to the relatively low OFC. He also exhibited a flat occiput, a long face, narrow bitemporal diameter, partial synophrys, mild
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hypertelorism, down-slanting palpebral fissures, ptosis, high and broad nasal bridge and a prominent nasal tip, high and narrow palate, large mouth with smooth, long and protruding philtrum, thin upper lip, full lower lip, and retrognathia (Figure 1 and Table 1). His fingers were long and slender, he had pectus excavatum and a scoliosis of about 20 degrees, pes transversoplanus, an extra nipple, adduction
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of fifth toes bilaterally and he wore insoles. Several teeth were extracted due to dental braces, and he had congenital lack of tooth number 25 and 34. A special exercise program was initiated to improve an oral muscular dysfunction. He had reduced fine motoric strength and tempo. The patient was
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hypermetric, had bilateral non-progressive sensorineural hearing loss, and was sensitive to light and loud sounds. He followed a special program at a regular high school, and worked part-time in an
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adapted work setting. He was good tempered with no behavior problems. He had polydipsia. Ultrasound examinations of kidneys and heart were normal. An electroencephalography (EEG) recording at standard conditions showed no pathology. Magnetic Resonance Imaging (MRI) caput was normal.
2. Methods of detection and confirmation Chromosome metaphase spreads taken from peripheral blood of the patient and his parents were analyzed by standard G-banding methods. Genomic DNA from leucocytes was analyzed by Array Comparative Genome Hybridization (aCGH) using Agilent 180K SurePrint G3 Human CGH (Agilent 3
ACCEPTED MANUSCRIPT Technologies, Santa Clara, CA, USA) according to the manufacturer's recommendations. Data were processed with Feature Extraction and DNA Analytics (Agilent Technologies). For confirmation, quantitative real-time PCR (q-PCR) analysis was performed on genomic DNA from the patient and his parents, as previously described [2]. Primer information is summarized in Supplementary Table 1. All
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human genome positions refer to the February 2009 human reference sequence (GRC37/hg19) produced by the Genome Reference Consortium. 3. Genomic rearrangement
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Karyotype analysis of the patient was normal, except for a benign variant: a fragile site at 12q with tentative breakpoints in q13.2 [3]. aCGH analysis of the patient’s DNA disclosed four imbalances: a
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230 kb deletion in chromosome 2p15 (chr2:61500346-61733075 bp) (Figure 2), two duplications in chromosome 9p24.3 of 490 kb (chr9:434742-928141 bp) and 410 kb (chr9:996466-1405431 bp), and a 660 kb duplication in 17q25.3 (chr17:79926623-80583456 bp). aCGH performed on DNA from the parents showed that the deletion in chr2p15 was de novo in the patient, while the duplications on
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chromosome 9p24.3 and chromosome 17q25.3 were maternally and paternally inherited, respectively. The chromosome 2p15 deletion affects the genes encoding Exportin 1 (XPO1) and Ubiquitin specific protease 34 (USP34).The deletion removed exons 5-26 including the 3’Untranslated Region (UTR) in
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XPO1, and exons 1-41 of the 80 exons in USP34 . In addition, the Small nucleolar RNA H/ACA box 70B gene, SNORA70B, was entirely deleted. The chromosome 2p15 deletion in the patient and the
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normal results for this region in the parents were verified by q-PCR (Supplementary Figure 1). The deletion is likely to be pathogenic because of its de novo origin and because it overlaps with a genomic region already emphasized to be critical in the 2p15p16.1 microdeletion syndrome. The significance of the SNORA70B deletion could not be assessed because it is a non-coding RNA of unknown function. Based on the expression pattern and function of XPO1 and USP34, we suggest that the partial deletion of these genes in our patient plays a crucial role in the aetiology of 2p15p16.1 microdeletion syndrome. 4. Discussion
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ACCEPTED MANUSCRIPT We report the case of a male patient who was first ascertained for language delay at the age of 4. Presently, at the age of 21 years, the patient functions in the area of mild ID/neurodevelopmental delay. He carries a de novo 2p15 deletion and three inherited duplications overlapping benign CNVs according to the Database of Genomic Variants (DGV, projects.tcag.ca/variation), which are not
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presently associated with disease. The duplication in 17q25.3 was inherited from the father, who has normal cognitive function, but a diagnosis of bipolar disorder. However, current knowledge does not link this duplication to psychiatric diseases. One published case of a patient with intellectual disability (DECIPHER ID 253687) (decipher.sanger.ac.uk/) described a de novo duplication in chr17:80067024-
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80561330 bp, in addition to several inherited CNVs. Another patient (ISCA ID nssv580950)
(iscaconsortium.org) who presented with global developmental delay, seizures, hypertelorism, and
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coloboma was found to carry a duplication in chr17:80036332-80525884 bp of unknown inheritance. The duplication in the latter patient was defined as having unknown clinical significance. With only two patients previously reported, and sparse information available, the functional consequence of the 17p25.3 duplication is uncertain. We suggest that this duplication, and the two other inherited
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duplications in our patient, might contribute to the clinical phenotype by cumulative CNV burden, resulting in the expression of clinical features with reduced penetrance or variable expressivity. The de novo 2p15 deletion in our patient overlaps structural variations annotated in the DGV.
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However, most of these were reported by a single study performed in three Asian populations [4]. In addition, the deletion in our patient overlaps with the known 2p15p16.1 microdeletion syndrome
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region, and the patient exhibits symptoms compatible with the phenotypic spectrum of this disease. Thus, the 2p15 deletion is likely the cause of his clinical presentation. The 2p15p16.1 microdeletion syndrome emerged as a distinct clinical entity when Rajcan-Separovic et al. [5] described two patients affected by moderate to severe ID, autism spectrum disorders (ASDs) or autistic features, microcephaly with cortical dysplasia/pachygyria, optic nerve hypoplasia, ptosis, shortened palpebral fissures, widened innercanthal distance, broadened nasal root and tip, everted lower lip, digital camptodactyly, renal anomalies and spasticity of the lower limbs. With the present report, 16 patients carrying a deletion within 2p15p16.1 are reported in the literature [5-15], [16] 5
ACCEPTED MANUSCRIPT (Patient 2). We compared the clinical presentation of our patient to 10 of those patients [5-9,12-14] while we excluded five patients ([10,11,15], [14] (Patient 1), [16] (Patient 2), due to lack of overlap between their genomic imbalance and that found in our patient (Table 1 and Figure 3). Five patients carrying overlapping deletions have been reported in DECIPHER (search done 20.11.2013). We
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included in the comparison three DECIPHER patients (ID 2172, ID 260600, ID 2855) (Table 1 and Figure 3), while we excluded two patients (Patient ID 1665 presenting with ID, ASD and 1666
presenting with ID, ASD and heterotopia), due to limited clinical information. ID/neurodevelopmental delay was the only clinical feature consistently diagnosed in all 14 patients compared. With the
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exception of our patient, who has a mild ID, the degree of ID ranges between moderate and severe in the other patients. It should be noted that the level of ID was not reported in any of the DECIPHER
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patients. A distinctive cranio-facial appearance appears to be common among the patients in our comparison group. Microcephaly, for example, was described in nine of the 14 patients, while the present patient exhibited an OFC measurement in the 10th centile, and Patient 2 from Piccione et al. [14] had an OFC < 10th centile. Of all the patients, only the one described by Chabchoub et al. [6]
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exhibited a relatively large head, with an OFC falling in the 75th-90th centile. Narrowing of the bitemporal diameter was seen in nine of the 14 patients. The facial dysmorphic traits seem to primarily affect the oral region and manifest with a smooth/long philtrum (10), a high narrow palate (nine),
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retrognathia or micrognathia (seven), a smooth/thin upper vermilion border (six), and everted lower lip (six). The oral area in our patient might be reminiscent of Williams syndrome or of fetal alcohol
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syndrome.
The patients also exhibited malformations affecting different organs, with a variety of brain defects, hypogonadism, genital anomalies and camptodactyly being the most frequent. None of those congenital defects were present in our patient. On the contrary, the malformations in our patient affect mainly the thorax, in the form of scoliosis, pectus excavatum and extra nipple. The patient described by Chabchoub et al. [6] and DECIPHER Patient ID 2172 exhibited both scoliosis and pectus excavatum, while an extra nipple was found in Patient 2 reported by Rajcan-Separovic [5]. Since
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ACCEPTED MANUSCRIPT scoliosis and pectus excavatum were only reported in the three patients harboring the smallest deletion (Table 1), those findings are either incidental or manifest with incomplete penetrance. Huchtagowder et al. [12] suggested that the region chr2:60.5-61.6 Mb, which contains 10 genes, is critical for 2p15p16.1 microdeletion syndrome, and proposed that the deletion of XPO1 might explain
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the multiple organ anomalies found in several patients haploinsufficient for this region. XPO1, or Chromosome Region Maintenance 1 (CRM1), is evolutionary conserved with a 96.7% amino acid identity between human and Xenopus laevis [17]. XPO1 functions as a nucleocytoplasmatic transport
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receptor mediating nuclear export of proteins and RNAs [18]. XPO1 plays an essential role in
coordinating nuclear events, such as mitosis and transcriptional activation [18] with nuclear transport,
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and may also be involved in maintenance of higher order chromosome structure. Callanan et al. [17] suggested that XPO1 might be a key regulator in early embryogenesis in Xenopus, playing a role during development at the gastrula-neural transition (GNT). They also showed that XPO1 overexpression during early development affected normal development at the neurula stage. Liu et al. [19] found that the rs6735330 SNP in XPO1 was significantly associated with autism susceptibility.
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All of the patients in our comparison group are haploinsufficient for XPO1, but only two of them received a diagnosis of ASD [5].
USP34, encoding a deubiquitinating enzyme (DUB) that is expressed in brain at a low level, is also
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deleted in our patient. A recent study indicated that within the nucleus, USP34 stabilizes the level of
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axin and positively modulates the Wingless-type (WNT) signalling pathway [20,21]. This pathway plays an important role in several developmental processes, such as cell fate specification, cell migration and proliferation. The WNT family of secreted glycoproteins modulates cell behavior, including differentiation and proliferation, both during embryonic development and in tissue homeostasis in adults. When disrupted, these signaling pathways cause developmental defects or diseases, among them cancer, cell movement and polarity defects. Patients carrying deletions in 2p15p16.1 not overlapping the deletion found in our patient clearly suggest the existence of additional genes within 2p15p16.1, whose haploinsufficiency is likely
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ACCEPTED MANUSCRIPT pathogenic. Two patients presenting with ID and dysmorphic features carried a 2p15p16.1 deletion not overlapping the deletion in our patient [11], [14] (Patient 1). Their deletion included BCL11A, encoding a zinc finger protein expressed in fetal brain that may play an import role in controlling neuronal morphology [22]. In addition, a de novo mutation in BCL11A was identified in a patient with
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ASD [23], a clinical feature reported in some patients with the 2p15p16.1 deletion syndrome. Two more ID patients have been reported [10], [16] (Patient 2) who carry deletions more centromeric in 2p15p16.1 not overlapping to the one in our patient. These observations would suggest that additional
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critical subregions exist.
Genetic heterogeneity likely explains some of the phenotypic differences between patients harboring
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deletions of different subregions or deletion spanning more than one critical subregion. In addition to differences in deletion size and gene content, reduced penetrance and variable expressivity may contribute to the observed phenotypic heterogeneity. On the other side, core traits of this syndrome seem to consist of a distinctive cranio-facial appearance and intellectual disability, which is often severe. Our patient presents both features, although his ID is in the mild range. Since two protein
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coding genes, XPO1 and USP34, are deleted in our patient, we suggest that one or both are likely responsible for the distinctive cranio-facial appearance and contribute to the intellectual disability observed in 2p15p16.1 deletion syndrome. This assumption seems reasonable given the fundamental
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cellular processes and signaling pathways where XPO1 and USP34 are involved.
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5. Acknowledgements
We would like to thank the family for collaboration and contribution to this project. We are grateful to Dr. William Louch for language revision of the manuscript. This work was supported by a grant from the Southeastern Regional Health Authorities (project no 2011071), Ullevål University Hospital Research Fund (VIRUUS), and DM was supported by “Anders Jahres fond til vitenskapens fremme”. Figures and tables legend Figure 1 Picture A, B: frontal and profile pictures of the patient at age 20. Dysmorphic features are described in the text. C. Scoliosis, pectus excavatum and extra nipple. 8
ACCEPTED MANUSCRIPT Figure 2 Graphical output of the aCGH results in the patient (left) and his parents (middle and right), revealing a de novo deletion in 2p15 (chr2:61500346-61733075 bp, hg19) in the patient. Dots represent the oligos along the chromosome. The area highlighted in black indicates the extension of the minimal deletion.
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Figure 3 Screenshot from USCS genome browser (genome.ucsc.edu) showing the deletion in our patient (red bar) and in the patients previously reported in the literature (black bars).
Table 1 Clinical comparison of the 2p15p16.1 deletion syndrome patients with overlap with the
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deletion in our patient.
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Supplementary Figure 1 Verification of the chromosome 2p15 deletion by q-PCR. A primer pair designed within the chromosome region defined by the aCGH results (In deletion) was used in q-PCR experiment on the patient DNA, the parents and on control DNA, confirming the deletion in the proband and its de novo origin. Normal DNA levels were detected with the control primer pair (Control) in all samples.
PCR experiments.
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Supplementary Table 1 Names, sequences and amplicon positions of all primer pairs used in the q-
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Reference List
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[11] M. Hancarova, M. Simandlova, J. Drabova, K. Mannik, A. Kurg, and Z. Sedlacek, A patient with de novo 0.45Mb deletion of 2p16.1: The role of BCL11A, PAPOLG, REL, and FLJ16341 in the 2p15-p16.1 microdeletion syndrome, American Journal of Medical Genetics Part A 161A (2013) 865-870.
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[13] J.S. Liang, K. Shimojima, K. Ohno, C. Sugiura, Y. Une, K. Ohno, and T. Yamamoto, A newly recognised microdeletion syndrome of 2p15-16.1 manifesting moderate developmental delay, autistic behaviour, short stature, microcephaly, and dysmorphic features: a new patient with 3.2 Mb deletion, J. Med. Genet. 46 (2009) 645-647. [14] M. Piccione, E. Piro, F. Serraino, S. Cavani, R. Ciccone, M. Malacarne, M. Pierluigi, M. Vitaloni, O. Zuffardi, and G. Corsello, Interstitial deletion of chromosome 2p15-16.1: report of two patients and critical review of current genotype-phenotype correlation, Eur. J. Med. Genet. 55 (2012) 238-244. [15] P. Prontera, L. Bernardini, G. Stangoni, A. Capalbo, D. Rogaia, R. Romani, C. Ardisia, B. Dallapiccola, and E. Donti, Deletion 2p15-16.1 syndrome: case report and review, Am. J. Med. Genet. A 155A (2011) 2473-2478. [16] E. Wohlleber, M. Kirchhoff, A.M. Zink, M. Kreiss-Nachtsheim, A. Kuchler, B. Jepsen, S. Kjaergaard, and H. Engels, Clinical and molecular characterization of two patients with 10
ACCEPTED MANUSCRIPT overlapping de novo microdeletions in 2p14-p15 and mild mental retardation, European Journal of Medical Genetics 54 (2011) 67-72. [17] M. Callanan, N. Kudo, S. Gout, M. Brocard, M. Yoshida, S. Dimitrov, and S. Khochbin, Developmentally regulated activity of CRM1/XPO1 during early Xenopus embryogenesis, J. Cell Sci. 113 ( Pt 3) (2000) 451-459.
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[20] T.T. Lui, C. Lacroix, S.M. Ahmed, S.J. Goldenberg, C.A. Leach, A.M. Daulat, and S. Angers, The ubiquitin-specific protease USP34 regulates axin stability and Wnt/beta-catenin signaling, Mol. Cell Biol. 31 (2011) 2053-2065.
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[21] S.M.H. Sy, J. Jiang, W.S. O, Y.Q. Deng, and M.S.Y. Huen, The ubiquitin specific protease USP34 promotes ubiquitin signaling at DNA double-strand breaks, Nucleic Acids Research 41 (2013) 8572-8580. [22] T.Y. Kuo, C.J. Hong, and Y.P. Hsueh, Bcl11A/CTIP1 regulates expression of DCC and MAP1b in control of axon branching and dendrite outgrowth, Mol. Cell Neurosci. 42 (2009) 195-207.
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[23] I. Iossifov, M. Ronemus, D. Levy, Z.H. Wang, I. Hakker, J. Rosenbaum, B. Yamrom, Y.H. Lee, G. Narzisi, A. Leotta, J. Kendall, E. Grabowska, B.C. Ma, S. Marks, L. Rodgers, A. Stepansky, J. Troge, P. Andrews, M. Bekritsky, K. Pradhan, E. Ghiban, M. Kramer, J. Parla, R. Demeter, L.L. Fulton, R.S. Fulton, V.J. Magrini, K. Ye, J.C. Darnell, R.B. Darnell, E.R. Mardis, R.K. Wilson, M.C. Schatz, W.R. McCombie, and M. Wigler, De Novo Gene Disruptions in Children on the Autistic Spectrum, Neuron 74 (2012) 285-299.
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Hucthagowder 2012
Piccione 2012 P2
Liang 2009
deLeeuw 2008
Decipher P ID 2855
Rajcan-Separovic 2007 P1
Rajcan-Separovic 2007 P2
Florisson 2012 P1
Florisson 2012 P2
570 kb 16 y -
980 kb nr nr nr
2.5 Mb 2y + +
2.5 Mb 7m + -
3.2 Mb 4.5 y + +
3.35 Mb 4y + +
3.9 Mb 32 y +
4.5 Mb
16 ya nr -
8 ya nr nr
4.5 Mb 8y + -
5.7 Mb 6y +
6.8 Mb 4y nr -
6.9 Mb 13 y nr -
5 5
-
nr
-
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+
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9
+/+/+
nr
+/-/+
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-/+/-
+/+/-
nr/-/nr
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-/+/+
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-/+/-
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-/+/-
-/+/-
3/9/3
+ + + + -
nr nr nr nr nr nr nr nr nr
nr nr + nr nr + nr nr
nr + nr nr nr nr nr + nr
+ + + nr +
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nr nr nr nr nr nr nr nr
+ + nr + + + nr nr
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4 5 4 3 5 8 9 4 6
+ +
nr nr
+ +
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+ -
+ nr
+ -
+ nr
+ +
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+ -
+ -
12 5
+ + + +
nr nr nr + +
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+ + nr nr nr
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Kidney anomaly Hypogonadism and/or genital malformations NEUROLOGICAL Structural brain anomalies Nevrodevelopmental delay
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GENITO-URINARY SYSTEM
Legend: +, feature present; -, examined and feature absent; Bit.narr, bitemporal narrowing; IUGR, intra uterin growth retardation; m, months; nr, not recorded; P, patient; y, years. a
age at first examination
Total (n=14)
Decipher P ID 260600
781 kb
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Chabchoub 2008
230 kb 20 y -
Felix 2010
Decipher P ID 2172
DELETION AGE AT LAST EXAMINATION IUGR SHORT STATURE CAPUT Microcephaly FACIAL FEATURES Long face/Bit.narr./High-sloping forehead Eyes Strabism Astigmatism or hypermetropia or nystagmus Optic nerve hypoplasia Downslanting palpebral fissures Short palpebral fissures Ptosis Increased interchantal distance and/or telechantus Hypertelorism Epichantal folds Nose High/broad nasal bridge Prominent nasal tip Oral region Smooth/long philtrum Smooth/thin upper vermilion border Everted lower lip Retrognathia/micrognathia High narrow palate Ears Hearing loss THORAX Increased internipple distance Extra nipple Pectus excavatum Scoliosis Cardiac malformtion HANDS/FEET Camptodactyly
Present patient
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ACCEPTED MANUSCRIPT Supplementary Table 1 Primer pairs used in q-PCR experiments Forward primer sequence TGTGACCTGTAGCGACCATC TGAGGTCAAAGGGAAGTTGC TTTTGGTAGCCCAAATGTCC
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Primer pair Control_F+R In deletion_F+R Endogenous control_F+R
ACCEPTED MANUSCRIPT Amplicon position (hg19) chr15:32272607-32272702 chr2:61667400-61667486 chr10:92633968-92634064
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Reverse primer sequence GAAATCCATCCCATCCACAC CCTCCTGATGTCCTGTCTGG CTGCACACCATCGACAGACT
S1769-7212(14)00131-1
DOI:
10.1016/j.ejmg.2014.05.008
Reference:
EJMG 2948
To appear in:
European Journal of Medical Genetics
Received Date: 13 January 2014 Accepted Date: 28 May 2014
Please cite this article as: M. Fannemel, T. Barøy, A. Holmgren, O.K. Rødningen, T.M. Haugsand, B. Hansen, E. Frengen, D. Misceo, Haploinsufficiency of XP01 and USP34 by a de novo 230 kb deletion in 2p15, in a patient with mild intellectual disability and cranio-facial dysmorphisms, European Journal of Medical Genetics (2014), doi: 10.1016/j.ejmg.2014.05.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Haploinsufficiency of XP01 and USP34 by a de novo 230 kb deletion in 2p15, in a patient with mild intellectual disability and cranio-facial dysmorphisms Running title: A de novo 230 kb deletion in 2p15 Madeleine Fannemel1, Tuva Barøy1, Asbjørn Holmgren1, Olaug K. Rødningen1, Trine M. Haugsand2,
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Børre Hansen2, Eirik Frengen1, Doriana Misceo1§
Department of Medical Genetics, University of Oslo and Oslo University Hospital, Oslo, Norway
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Department for adult habilitation, Akershus University Hospital, Oslo, Norway
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MF: [email protected] TB: [email protected]
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E-mail addresses:
AH: [email protected]
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OKR: [email protected]
TMH: [email protected]
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BH: [email protected]
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EF: [email protected] DM: [email protected]
§ To whom correspondence should be addressed: Doriana Misceo, PhD, Department of Medical Genetics, University of Oslo, P.O. Box 1036, Blindern, N-0315 Oslo, Norway. [email protected]; telephone: +47 22116228, fax: +47 22119899 Disclosure statement No competing financial interests exist.
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ACCEPTED MANUSCRIPT Abstract 2p15p16.1-deletion syndrome was first described in 2007 based on the clinical presentation of two patients. The syndrome is characterized by intellectual disability, autism spectrum disorders, microcephaly, dysmorphic facial features and a variety of congenital organ defects. The precise
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genotype-phenotype correlation in 2p15- deletion syndrome is not understood. However, greater
insight can be obtained by thorough clinical investigation of patients carrying deletions, especially those of small size. We report a 21-year-old male patient with features overlapping the clinical
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spectrum of the 2p15p16.1 deletion syndrome, such as intellectual disability, dysmorphic facial
features, and congenital defects. He carried a 230 kb de novo deletion (chr2:61500346-61733075 bp,
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hg19), which affects the genes USP34, SNORA70B and XPO1. While there is a lack of functional data on SNORA70B, the involvement of USP34 and XPO1 in the regulation of fundamental developmental processes is well known. We suggest that haploinsuffiency of one or both of these genes is likely to be responsible for the disease in our patient.
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Keywords: congenital anomalies, dysmorphic features, intellectual disability, USP34, XPO1 or CRM1, 2p15p16.1 deletion syndrome. 1. Clinical description
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The patient is a 21-year-old male, single child of a non-consanguineous Norwegian couple. His father was diagnosed with bipolar disorder. Early pregnancy was complicated by small bleedings lasting
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about one week and maternal hyperthyreosis. No drug or alcohol exposure during the pregnancy was reported by the mother. The patient was born at term with a weight of 3240 g (10th-25th centile), a length of 52 cm (75th centile), while occipitofrontal circumference (OFC) was not recorded (Norwegian growth standards are in use [1]). Apgar score was normal. Amniotic fluid was discolored. He started to walk at the age of 13-14 months and spoke his first word at 1 year. There were concerns about his language development from the age of 2. At the age of 4 he scored more than 3 standard deviations (SD) below the age mean in all fields on McCarthy’s scales of children’s ability (MCSA). He was repeatedly evaluated during childhood and he followed a special educational program. An
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ACCEPTED MANUSCRIPT extensive panel of cognitive and behavioral tests were performed at the age of 20 years. The results were consistent with mild intellectual disability (ID). Interestingly, the patient scored 63 on the Wechsler adult intelligence scale (WAIS IV), corresponding to a developmental age of 10-12 years (using a 95% confidence interval), but according to the Adaptive Behavior Assessment System
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(ABAS) he scored in the range of 7-8 years, which is more than 2 SD below the WAIS IV results. At 20 years of age he sought a psychologist due to anxiety, obsessive compulsive actions and ideas. He received no medication for this. At his most recent examination at the age of 21 his height was 174 cm (10th-25th centile), weight 52 kg (10th centile), and OFC 55.2 cm (10th centile). Facial dysmorphic
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features included a high, narrow and sloping forehead, possibly due to the relatively low OFC. He also exhibited a flat occiput, a long face, narrow bitemporal diameter, partial synophrys, mild
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hypertelorism, down-slanting palpebral fissures, ptosis, high and broad nasal bridge and a prominent nasal tip, high and narrow palate, large mouth with smooth, long and protruding philtrum, thin upper lip, full lower lip, and retrognathia (Figure 1 and Table 1). His fingers were long and slender, he had pectus excavatum and a scoliosis of about 20 degrees, pes transversoplanus, an extra nipple, adduction
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of fifth toes bilaterally and he wore insoles. Several teeth were extracted due to dental braces, and he had congenital lack of tooth number 25 and 34. A special exercise program was initiated to improve an oral muscular dysfunction. He had reduced fine motoric strength and tempo. The patient was
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hypermetric, had bilateral non-progressive sensorineural hearing loss, and was sensitive to light and loud sounds. He followed a special program at a regular high school, and worked part-time in an
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adapted work setting. He was good tempered with no behavior problems. He had polydipsia. Ultrasound examinations of kidneys and heart were normal. An electroencephalography (EEG) recording at standard conditions showed no pathology. Magnetic Resonance Imaging (MRI) caput was normal.
2. Methods of detection and confirmation Chromosome metaphase spreads taken from peripheral blood of the patient and his parents were analyzed by standard G-banding methods. Genomic DNA from leucocytes was analyzed by Array Comparative Genome Hybridization (aCGH) using Agilent 180K SurePrint G3 Human CGH (Agilent 3
ACCEPTED MANUSCRIPT Technologies, Santa Clara, CA, USA) according to the manufacturer's recommendations. Data were processed with Feature Extraction and DNA Analytics (Agilent Technologies). For confirmation, quantitative real-time PCR (q-PCR) analysis was performed on genomic DNA from the patient and his parents, as previously described [2]. Primer information is summarized in Supplementary Table 1. All
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human genome positions refer to the February 2009 human reference sequence (GRC37/hg19) produced by the Genome Reference Consortium. 3. Genomic rearrangement
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Karyotype analysis of the patient was normal, except for a benign variant: a fragile site at 12q with tentative breakpoints in q13.2 [3]. aCGH analysis of the patient’s DNA disclosed four imbalances: a
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230 kb deletion in chromosome 2p15 (chr2:61500346-61733075 bp) (Figure 2), two duplications in chromosome 9p24.3 of 490 kb (chr9:434742-928141 bp) and 410 kb (chr9:996466-1405431 bp), and a 660 kb duplication in 17q25.3 (chr17:79926623-80583456 bp). aCGH performed on DNA from the parents showed that the deletion in chr2p15 was de novo in the patient, while the duplications on
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chromosome 9p24.3 and chromosome 17q25.3 were maternally and paternally inherited, respectively. The chromosome 2p15 deletion affects the genes encoding Exportin 1 (XPO1) and Ubiquitin specific protease 34 (USP34).The deletion removed exons 5-26 including the 3’Untranslated Region (UTR) in
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XPO1, and exons 1-41 of the 80 exons in USP34 . In addition, the Small nucleolar RNA H/ACA box 70B gene, SNORA70B, was entirely deleted. The chromosome 2p15 deletion in the patient and the
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normal results for this region in the parents were verified by q-PCR (Supplementary Figure 1). The deletion is likely to be pathogenic because of its de novo origin and because it overlaps with a genomic region already emphasized to be critical in the 2p15p16.1 microdeletion syndrome. The significance of the SNORA70B deletion could not be assessed because it is a non-coding RNA of unknown function. Based on the expression pattern and function of XPO1 and USP34, we suggest that the partial deletion of these genes in our patient plays a crucial role in the aetiology of 2p15p16.1 microdeletion syndrome. 4. Discussion
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ACCEPTED MANUSCRIPT We report the case of a male patient who was first ascertained for language delay at the age of 4. Presently, at the age of 21 years, the patient functions in the area of mild ID/neurodevelopmental delay. He carries a de novo 2p15 deletion and three inherited duplications overlapping benign CNVs according to the Database of Genomic Variants (DGV, projects.tcag.ca/variation), which are not
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presently associated with disease. The duplication in 17q25.3 was inherited from the father, who has normal cognitive function, but a diagnosis of bipolar disorder. However, current knowledge does not link this duplication to psychiatric diseases. One published case of a patient with intellectual disability (DECIPHER ID 253687) (decipher.sanger.ac.uk/) described a de novo duplication in chr17:80067024-
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80561330 bp, in addition to several inherited CNVs. Another patient (ISCA ID nssv580950)
(iscaconsortium.org) who presented with global developmental delay, seizures, hypertelorism, and
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coloboma was found to carry a duplication in chr17:80036332-80525884 bp of unknown inheritance. The duplication in the latter patient was defined as having unknown clinical significance. With only two patients previously reported, and sparse information available, the functional consequence of the 17p25.3 duplication is uncertain. We suggest that this duplication, and the two other inherited
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duplications in our patient, might contribute to the clinical phenotype by cumulative CNV burden, resulting in the expression of clinical features with reduced penetrance or variable expressivity. The de novo 2p15 deletion in our patient overlaps structural variations annotated in the DGV.
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However, most of these were reported by a single study performed in three Asian populations [4]. In addition, the deletion in our patient overlaps with the known 2p15p16.1 microdeletion syndrome
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region, and the patient exhibits symptoms compatible with the phenotypic spectrum of this disease. Thus, the 2p15 deletion is likely the cause of his clinical presentation. The 2p15p16.1 microdeletion syndrome emerged as a distinct clinical entity when Rajcan-Separovic et al. [5] described two patients affected by moderate to severe ID, autism spectrum disorders (ASDs) or autistic features, microcephaly with cortical dysplasia/pachygyria, optic nerve hypoplasia, ptosis, shortened palpebral fissures, widened innercanthal distance, broadened nasal root and tip, everted lower lip, digital camptodactyly, renal anomalies and spasticity of the lower limbs. With the present report, 16 patients carrying a deletion within 2p15p16.1 are reported in the literature [5-15], [16] 5
ACCEPTED MANUSCRIPT (Patient 2). We compared the clinical presentation of our patient to 10 of those patients [5-9,12-14] while we excluded five patients ([10,11,15], [14] (Patient 1), [16] (Patient 2), due to lack of overlap between their genomic imbalance and that found in our patient (Table 1 and Figure 3). Five patients carrying overlapping deletions have been reported in DECIPHER (search done 20.11.2013). We
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included in the comparison three DECIPHER patients (ID 2172, ID 260600, ID 2855) (Table 1 and Figure 3), while we excluded two patients (Patient ID 1665 presenting with ID, ASD and 1666
presenting with ID, ASD and heterotopia), due to limited clinical information. ID/neurodevelopmental delay was the only clinical feature consistently diagnosed in all 14 patients compared. With the
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exception of our patient, who has a mild ID, the degree of ID ranges between moderate and severe in the other patients. It should be noted that the level of ID was not reported in any of the DECIPHER
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patients. A distinctive cranio-facial appearance appears to be common among the patients in our comparison group. Microcephaly, for example, was described in nine of the 14 patients, while the present patient exhibited an OFC measurement in the 10th centile, and Patient 2 from Piccione et al. [14] had an OFC < 10th centile. Of all the patients, only the one described by Chabchoub et al. [6]
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exhibited a relatively large head, with an OFC falling in the 75th-90th centile. Narrowing of the bitemporal diameter was seen in nine of the 14 patients. The facial dysmorphic traits seem to primarily affect the oral region and manifest with a smooth/long philtrum (10), a high narrow palate (nine),
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retrognathia or micrognathia (seven), a smooth/thin upper vermilion border (six), and everted lower lip (six). The oral area in our patient might be reminiscent of Williams syndrome or of fetal alcohol
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syndrome.
The patients also exhibited malformations affecting different organs, with a variety of brain defects, hypogonadism, genital anomalies and camptodactyly being the most frequent. None of those congenital defects were present in our patient. On the contrary, the malformations in our patient affect mainly the thorax, in the form of scoliosis, pectus excavatum and extra nipple. The patient described by Chabchoub et al. [6] and DECIPHER Patient ID 2172 exhibited both scoliosis and pectus excavatum, while an extra nipple was found in Patient 2 reported by Rajcan-Separovic [5]. Since
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ACCEPTED MANUSCRIPT scoliosis and pectus excavatum were only reported in the three patients harboring the smallest deletion (Table 1), those findings are either incidental or manifest with incomplete penetrance. Huchtagowder et al. [12] suggested that the region chr2:60.5-61.6 Mb, which contains 10 genes, is critical for 2p15p16.1 microdeletion syndrome, and proposed that the deletion of XPO1 might explain
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the multiple organ anomalies found in several patients haploinsufficient for this region. XPO1, or Chromosome Region Maintenance 1 (CRM1), is evolutionary conserved with a 96.7% amino acid identity between human and Xenopus laevis [17]. XPO1 functions as a nucleocytoplasmatic transport
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receptor mediating nuclear export of proteins and RNAs [18]. XPO1 plays an essential role in
coordinating nuclear events, such as mitosis and transcriptional activation [18] with nuclear transport,
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and may also be involved in maintenance of higher order chromosome structure. Callanan et al. [17] suggested that XPO1 might be a key regulator in early embryogenesis in Xenopus, playing a role during development at the gastrula-neural transition (GNT). They also showed that XPO1 overexpression during early development affected normal development at the neurula stage. Liu et al. [19] found that the rs6735330 SNP in XPO1 was significantly associated with autism susceptibility.
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All of the patients in our comparison group are haploinsufficient for XPO1, but only two of them received a diagnosis of ASD [5].
USP34, encoding a deubiquitinating enzyme (DUB) that is expressed in brain at a low level, is also
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deleted in our patient. A recent study indicated that within the nucleus, USP34 stabilizes the level of
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axin and positively modulates the Wingless-type (WNT) signalling pathway [20,21]. This pathway plays an important role in several developmental processes, such as cell fate specification, cell migration and proliferation. The WNT family of secreted glycoproteins modulates cell behavior, including differentiation and proliferation, both during embryonic development and in tissue homeostasis in adults. When disrupted, these signaling pathways cause developmental defects or diseases, among them cancer, cell movement and polarity defects. Patients carrying deletions in 2p15p16.1 not overlapping the deletion found in our patient clearly suggest the existence of additional genes within 2p15p16.1, whose haploinsufficiency is likely
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ACCEPTED MANUSCRIPT pathogenic. Two patients presenting with ID and dysmorphic features carried a 2p15p16.1 deletion not overlapping the deletion in our patient [11], [14] (Patient 1). Their deletion included BCL11A, encoding a zinc finger protein expressed in fetal brain that may play an import role in controlling neuronal morphology [22]. In addition, a de novo mutation in BCL11A was identified in a patient with
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ASD [23], a clinical feature reported in some patients with the 2p15p16.1 deletion syndrome. Two more ID patients have been reported [10], [16] (Patient 2) who carry deletions more centromeric in 2p15p16.1 not overlapping to the one in our patient. These observations would suggest that additional
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critical subregions exist.
Genetic heterogeneity likely explains some of the phenotypic differences between patients harboring
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deletions of different subregions or deletion spanning more than one critical subregion. In addition to differences in deletion size and gene content, reduced penetrance and variable expressivity may contribute to the observed phenotypic heterogeneity. On the other side, core traits of this syndrome seem to consist of a distinctive cranio-facial appearance and intellectual disability, which is often severe. Our patient presents both features, although his ID is in the mild range. Since two protein
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coding genes, XPO1 and USP34, are deleted in our patient, we suggest that one or both are likely responsible for the distinctive cranio-facial appearance and contribute to the intellectual disability observed in 2p15p16.1 deletion syndrome. This assumption seems reasonable given the fundamental
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cellular processes and signaling pathways where XPO1 and USP34 are involved.
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5. Acknowledgements
We would like to thank the family for collaboration and contribution to this project. We are grateful to Dr. William Louch for language revision of the manuscript. This work was supported by a grant from the Southeastern Regional Health Authorities (project no 2011071), Ullevål University Hospital Research Fund (VIRUUS), and DM was supported by “Anders Jahres fond til vitenskapens fremme”. Figures and tables legend Figure 1 Picture A, B: frontal and profile pictures of the patient at age 20. Dysmorphic features are described in the text. C. Scoliosis, pectus excavatum and extra nipple. 8
ACCEPTED MANUSCRIPT Figure 2 Graphical output of the aCGH results in the patient (left) and his parents (middle and right), revealing a de novo deletion in 2p15 (chr2:61500346-61733075 bp, hg19) in the patient. Dots represent the oligos along the chromosome. The area highlighted in black indicates the extension of the minimal deletion.
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Figure 3 Screenshot from USCS genome browser (genome.ucsc.edu) showing the deletion in our patient (red bar) and in the patients previously reported in the literature (black bars).
Table 1 Clinical comparison of the 2p15p16.1 deletion syndrome patients with overlap with the
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deletion in our patient.
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Supplementary Figure 1 Verification of the chromosome 2p15 deletion by q-PCR. A primer pair designed within the chromosome region defined by the aCGH results (In deletion) was used in q-PCR experiment on the patient DNA, the parents and on control DNA, confirming the deletion in the proband and its de novo origin. Normal DNA levels were detected with the control primer pair (Control) in all samples.
PCR experiments.
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Supplementary Table 1 Names, sequences and amplicon positions of all primer pairs used in the q-
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Reference List
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[1] P.B. Juliusson, M. Roelants, G.E. Eide, D. Moster, A. Juul, R. Hauspie, P.E. Waaler, and R. Bjerknes, [Growth references for Norwegian children], Tidsskr. Nor Laegeforen. 129 (2009) 281-286. [2] K. Floor, T. Baroy, D. Misceo, O.J. Kanavin, M. Fannemel, and E. Frengen, A 1 Mb de novo deletion within 11q13.1q13.2 in a boy with mild intellectual disability and minor dysmorphic features, European Journal of Medical Genetics 55 (2012) 695-699. [3] T. Lukusa and J.P. Fryns, Human chromosome fragility, Biochim. Biophys. Acta 1779 (2008) 316. [4] H.Y. Xu, W.T. Poh, X.L. Sim, R.T.H. Ong, C. Suo, W.T. Tay, C.C. Khor, M. Seielstad, J.J. Liu, T. Aung, E.S. Tai, T.Y. Wong, K.S. Chia, and Y.Y. Teo, SgD-CNV, a database for common and rare copy number variants in three Asian populations, Human Mutation 32 (2011) 1341-1349.
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ACCEPTED MANUSCRIPT [5] E. Rajcan-Separovic, C. Harvard, X. Liu, B. McGillivray, J.G. Hall, Y. Qiao, J. Hurlburt, J. Hildebrand, E.C. Mickelson, J.J. Holden, and M.E. Lewis, Clinical and molecular cytogenetic characterisation of a newly recognised microdeletion syndrome involving 2p15-16.1, J. Med. Genet. 44 (2007) 269-276. [6] E. Chabchoub, J.R. Vermeesch, R.T. de, C.P. de, and J.P. Fryns, The facial dysmorphy in the newly recognised microdeletion 2p15-p16.1 refined to a 570 kb region in 2p15, J. Med. Genet. 45 (2008) 189-192.
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[8] T.M. Felix, A.L. Petrin, M.T. Sanseverino, and J.C. Murray, Further characterization of microdeletion syndrome involving 2p15-p16.1, Am. J. Med. Genet. A 152A (2010) 2604-2608.
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[9] J.M.G. Florisson, I.M.J. Mathijssen, B. Dumee, J.A.M. Hoogeboom, P.J. Poddighe, B.A. Oostra, J.P. Frijns, L. Koster, A. de Klein, B. Eussen, B.B.A. de Vries, S. Swagemakers, P.J. van der Spek, and A.J.M.H. Verkerk, Complex craniosynostosis is associated with the 2p15p16.1 microdeletion syndrome, American Journal of Medical Genetics Part A 161A (2013) 244-253. [10] M. Hancarova, S. Vejvalkova, M. Trkova, J. Drabova, A. Dleskova, M. Vlckova, and Z. Sedlacek, Identification of a patient with intellectual disability and de novo 3.7Mb deletion supports the existence of a novel microdeletion syndrome in 2p14-p15, Gene 516 (2013) 158-161.
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[11] M. Hancarova, M. Simandlova, J. Drabova, K. Mannik, A. Kurg, and Z. Sedlacek, A patient with de novo 0.45Mb deletion of 2p16.1: The role of BCL11A, PAPOLG, REL, and FLJ16341 in the 2p15-p16.1 microdeletion syndrome, American Journal of Medical Genetics Part A 161A (2013) 865-870.
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[12] V. Hucthagowder, T.C. Liu, A.R. Paciorkowski, L.L. Thio, M.S. Keller, C.D. Anderson, T. Herman, L.P. Dehner, D.K. Grange, and S. Kulkarni, Chromosome 2p15p16.1 microdeletion syndrome: 2.5 Mb deletion in a patient with renal anomalies, intractable seizures and a choledochal cyst, Eur. J. Med. Genet. 55 (2012) 485-489.
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[13] J.S. Liang, K. Shimojima, K. Ohno, C. Sugiura, Y. Une, K. Ohno, and T. Yamamoto, A newly recognised microdeletion syndrome of 2p15-16.1 manifesting moderate developmental delay, autistic behaviour, short stature, microcephaly, and dysmorphic features: a new patient with 3.2 Mb deletion, J. Med. Genet. 46 (2009) 645-647. [14] M. Piccione, E. Piro, F. Serraino, S. Cavani, R. Ciccone, M. Malacarne, M. Pierluigi, M. Vitaloni, O. Zuffardi, and G. Corsello, Interstitial deletion of chromosome 2p15-16.1: report of two patients and critical review of current genotype-phenotype correlation, Eur. J. Med. Genet. 55 (2012) 238-244. [15] P. Prontera, L. Bernardini, G. Stangoni, A. Capalbo, D. Rogaia, R. Romani, C. Ardisia, B. Dallapiccola, and E. Donti, Deletion 2p15-16.1 syndrome: case report and review, Am. J. Med. Genet. A 155A (2011) 2473-2478. [16] E. Wohlleber, M. Kirchhoff, A.M. Zink, M. Kreiss-Nachtsheim, A. Kuchler, B. Jepsen, S. Kjaergaard, and H. Engels, Clinical and molecular characterization of two patients with 10
ACCEPTED MANUSCRIPT overlapping de novo microdeletions in 2p14-p15 and mild mental retardation, European Journal of Medical Genetics 54 (2011) 67-72. [17] M. Callanan, N. Kudo, S. Gout, M. Brocard, M. Yoshida, S. Dimitrov, and S. Khochbin, Developmentally regulated activity of CRM1/XPO1 during early Xenopus embryogenesis, J. Cell Sci. 113 ( Pt 3) (2000) 451-459.
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[18] S. Hutten and R.H. Kehlenbach, CRM1-mediated nuclear export: to the pore and beyond, Trends Cell Biol. 17 (2007) 193-201. [19] X. Liu, P. Malenfant, C. Reesor, A. Lee, M.L. Hudson, C. Harvard, Y. Qiao, A.M. Persico, I.L. Cohen, A.E. Chudley, C. Forster-Gibson, E. Rajcan-Separovic, M.E. Lewis, and J.J. Holden, 2p15-p16.1 microdeletion syndrome: molecular characterization and association of the OTX1 and XPO1 genes with autism spectrum disorders, Eur. J. Hum. Genet. 19 (2011) 1264-1270.
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[20] T.T. Lui, C. Lacroix, S.M. Ahmed, S.J. Goldenberg, C.A. Leach, A.M. Daulat, and S. Angers, The ubiquitin-specific protease USP34 regulates axin stability and Wnt/beta-catenin signaling, Mol. Cell Biol. 31 (2011) 2053-2065.
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[21] S.M.H. Sy, J. Jiang, W.S. O, Y.Q. Deng, and M.S.Y. Huen, The ubiquitin specific protease USP34 promotes ubiquitin signaling at DNA double-strand breaks, Nucleic Acids Research 41 (2013) 8572-8580. [22] T.Y. Kuo, C.J. Hong, and Y.P. Hsueh, Bcl11A/CTIP1 regulates expression of DCC and MAP1b in control of axon branching and dendrite outgrowth, Mol. Cell Neurosci. 42 (2009) 195-207.
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[23] I. Iossifov, M. Ronemus, D. Levy, Z.H. Wang, I. Hakker, J. Rosenbaum, B. Yamrom, Y.H. Lee, G. Narzisi, A. Leotta, J. Kendall, E. Grabowska, B.C. Ma, S. Marks, L. Rodgers, A. Stepansky, J. Troge, P. Andrews, M. Bekritsky, K. Pradhan, E. Ghiban, M. Kramer, J. Parla, R. Demeter, L.L. Fulton, R.S. Fulton, V.J. Magrini, K. Ye, J.C. Darnell, R.B. Darnell, E.R. Mardis, R.K. Wilson, M.C. Schatz, W.R. McCombie, and M. Wigler, De Novo Gene Disruptions in Children on the Autistic Spectrum, Neuron 74 (2012) 285-299.
11
Hucthagowder 2012
Piccione 2012 P2
Liang 2009
deLeeuw 2008
Decipher P ID 2855
Rajcan-Separovic 2007 P1
Rajcan-Separovic 2007 P2
Florisson 2012 P1
Florisson 2012 P2
570 kb 16 y -
980 kb nr nr nr
2.5 Mb 2y + +
2.5 Mb 7m + -
3.2 Mb 4.5 y + +
3.35 Mb 4y + +
3.9 Mb 32 y +
4.5 Mb
16 ya nr -
8 ya nr nr
4.5 Mb 8y + -
5.7 Mb 6y +
6.8 Mb 4y nr -
6.9 Mb 13 y nr -
5 5
-
nr
-
nr
+
-
+
+
+
+
+
+
+
+
9
+/+/+
nr
+/-/+
nr
-/+/-
+/+/-
nr/-/nr
nr
-/+/+
-/+/-
-/+/-
-/+/-
-/+/-
-/+/-
3/9/3
+ + + + -
nr nr nr nr nr nr nr nr nr
nr nr + nr nr + nr nr
nr + nr nr nr nr nr + nr
+ + + nr +
nr nr +
nr + + + + + +
+ nr + + + nr
+ + + + + + + +
nr nr nr nr nr nr nr nr
+ + nr + + + nr nr
+ + nr + + + nr nr
+ nr + + +
+ + +
4 5 4 3 5 8 9 4 6
+ +
nr nr
+ +
+ nr
+ +
nr
+ -
+ nr
+ -
+ nr
+ +
+ +
+ -
+ -
12 5
+ + + +
nr nr nr + +
nr + + nr +
+ + nr nr nr
nr nr + + +
+
nr
-
nr
+ + + -
nr nr + + +
nr nr + + +
nr nr nr nr nr
-
nr
nr
-
nr +
nr +
Mild
nr +
Moderate
Kidney anomaly Hypogonadism and/or genital malformations NEUROLOGICAL Structural brain anomalies Nevrodevelopmental delay
SC
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TE D + + -
+ nr nr + +
+ + + + +
nr nr nr + nr
+ + + nr +
+ + + nr +
+ -
+ -
10 6 6 7 9
-
+
-
-
-
nr
-
+
-
+
4
nr nr nr nr nr
nr nr nr nr -
nr nr nr
+ nr nr nr -
+ nr nr nr
nr nr nr nr nr
+ nr nr nr nr
+ + nr nr nr
nr nr nr
nr nr nr
4 2 3 3 2
nr
nr
+
-
+
-
nr
+
+
+
+
6
+ +
+ nr
nr +
-
nr nr
+ +
nr nr
+ nr
+ +
nr -
nr -
5 6
+ +
+ Severe
+ Severe
Severe
Moderate
nr Moderate to severe
nr +
+ Moderate
+ Moderate
+ Moderate
nr Severe
6 14
EP
+ + nr +
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GENITO-URINARY SYSTEM
Legend: +, feature present; -, examined and feature absent; Bit.narr, bitemporal narrowing; IUGR, intra uterin growth retardation; m, months; nr, not recorded; P, patient; y, years. a
age at first examination
Total (n=14)
Decipher P ID 260600
781 kb
RI PT
Chabchoub 2008
230 kb 20 y -
Felix 2010
Decipher P ID 2172
DELETION AGE AT LAST EXAMINATION IUGR SHORT STATURE CAPUT Microcephaly FACIAL FEATURES Long face/Bit.narr./High-sloping forehead Eyes Strabism Astigmatism or hypermetropia or nystagmus Optic nerve hypoplasia Downslanting palpebral fissures Short palpebral fissures Ptosis Increased interchantal distance and/or telechantus Hypertelorism Epichantal folds Nose High/broad nasal bridge Prominent nasal tip Oral region Smooth/long philtrum Smooth/thin upper vermilion border Everted lower lip Retrognathia/micrognathia High narrow palate Ears Hearing loss THORAX Increased internipple distance Extra nipple Pectus excavatum Scoliosis Cardiac malformtion HANDS/FEET Camptodactyly
Present patient
ACCEPTED MANUSCRIPT
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EP
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M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Supplementary Table 1 Primer pairs used in q-PCR experiments Forward primer sequence TGTGACCTGTAGCGACCATC TGAGGTCAAAGGGAAGTTGC TTTTGGTAGCCCAAATGTCC
AC C
EP
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M AN U
SC
RI PT
Primer pair Control_F+R In deletion_F+R Endogenous control_F+R
ACCEPTED MANUSCRIPT Amplicon position (hg19) chr15:32272607-32272702 chr2:61667400-61667486 chr10:92633968-92634064
AC C
EP
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M AN U
SC
RI PT
Reverse primer sequence GAAATCCATCCCATCCACAC CCTCCTGATGTCCTGTCTGG CTGCACACCATCGACAGACT
