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Phenotypic and functional consequences of haploinsufficiency of genes from exocyst and retinoic acid… Wen, Jiadi; Lopes, Fátima; Soares, Gabriela; Farrell, Sandra A; Nelson, Cara; Qiao, Ying; Martell, Sally; Badukke, Chansonette; Bessa, Carlos; Ylstra, Bauke; Lewis, Suzanne; Isoherranen, Nina; Maciel, Patricia; Rajcan-Separovic, Evica Jul 10, 2013

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RESEARCH Open AccessPhenotypic and functional consequences ofhaploinsufficiency of genes from exocyst andretinoic acid pathway due to a recurrentmicrodeletion of 2p13.2Jiadi Wen1†, Fátima Lopes2,3†, Gabriela Soares4, Sandra A Farrell5, Cara Nelson6, Ying Qiao1, Sally Martell1,Chansonette Badukke1, Carlos Bessa2,3, Bauke Ylstra7, Suzanne Lewis8, Nina Isoherranen6, Patricia Maciel2,3*and Evica Rajcan-Separovic1*AbstractBackground: Rare, recurrent genomic imbalances facilitate the association of genotype with abnormalities at the“whole body” level. However, at the cellular level, the functional consequences of recurrent genomic abnormalitiesand how they can be linked to the phenotype are much less investigated.Method and results: We report an example of a functional analysis of two genes from a new, overlappingmicrodeletion of 2p13.2 region (from 72,140,702-72,924,626). The subjects shared intellectual disability (ID), languagedelay, hyperactivity, facial asymmetry, ear malformations, and vertebral and/or craniofacial abnormalities. Theoverlapping region included two genes, EXOC6B and CYP26B1, which are involved in exocytosis/Notch signalingand retinoic acid (RA) metabolism, respectively, and are of critical importance for early morphogenesis, symmetry aswell as craniofacial, skeleton and brain development. The abnormal function of EXOC6B was documented in patientlymphoblasts by its reduced expression and with perturbed expression of Notch signaling pathway genes HES1 andRBPJ, previously noted to be the consequence of EXOC6B dysfunction in animal and cell line models. Similarly, thefunction of CYP26B1 was affected by the deletion since the retinoic acid induced expression of this gene in patientlymphoblasts was significantly lower compared to controls (8% of controls).Conclusion: Haploinsufficiency of CYP26B1 and EXOC6B genes involved in retinoic acid and exocyst/Notchsignaling pathways, respectively, has not been reported previously in humans. The developmental anomalies andphenotypic features of our subjects are in keeping with the dysfunction of these genes, considering their knownrole. Documenting their dysfunction at the cellular level in patient cells enhanced our understanding of biologicalprocesses which contribute to the clinical phenotype.Keywords: 2p13 deletion, EXOC6B, CYP26B1, Developmental delay, Cranial/skeletal anomalies* Correspondence:;†Equal contributors2Life and Health Sciences Research Institute (ICVS), School of Health Sciences,University of Minho, Braga, Portugal1Child and Family Research Institute, Department of Pathology, University ofBritish Columbia, Vancouver, BC, CanadaFull list of author information is available at the end of the article© 2013 Wen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (, which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Wen et al. Orphanet Journal of Rare Diseases 2013, 8:100 abnormalities involving the 2p13 chromo-somal region were detected previously in subjects withdevelopmental delay using traditional chromosome test-ing [1,2]. Abnormal features present in at least 50% ofcases included abnormal head size/shape, nose, ears,chest and vertebral, digital and genital anomalies. Theinvolvement of the CYP26B1 gene was suggested in asubject with a de novo inversion involving 2p13 and2q34, who had a Klippel-Fiel anomaly (vertebral fusionof cervical spine), psychomotor retardation, speech limi-tation, facial asymmetry, ear abnormalities, and scoliosis[3,4]. Disruption of EXOC6B and its fusion with TNS3from 7p12 due to a translocation t(2;7)(p13;p12), was con-sidered to be a possible cause of the intellectual disability,ADHD and congenital abnormalities (renal malformation,microcephaly, long bone diaphyseal broadening) in a sub-ject reported by Borsani et al. [5].CYP26B1 (cytochrome P450, family 26, subfamily B,polypeptide 1) is one of the three CYP26 gene isoforms(CYP26B1, CYP26A1 and CYP26C1) which encode thecytochrome-P450 enzymes that catabolize retinoic acid(RA) [6]. RA is the principal active metabolite of vitaminA and is an essential component of cell-cell signalingduring vertebrate organogenesis [7]. Too little or toomuch RA causes the human malformation syndrome as-sociated with vitamin A deficiency (VAD) or RAembryopathy, which includes craniofacial (e.g. ears, eyes,facial asymmetry), central nervous, musculoskeletal, andurogenital abnormalities [8-12]. Homozygous knockoutof CYP26B1 in animals has been associated with postna-tal mortality, abnormal craniofacial, limb and gonadaldevelopment [13,14], while a conditional deletion (con-trolled expression) of CYP26B1 resulted in a less severephenotype. The difference in the phenotypes was attrib-uted to differing levels of activated retinoid signaling[15]. Although deletions of the whole CYP26B1 genehave not yet been reported in humans, homozygouspoint mutations of CYP26B1 were reported in twofamilies, resulting in prenatal and early postnatal le-thality, skeletal and craniofacial abnormalities, fusionof long bones, calvarial bone hypoplasia and suturaldefects, resulting in craniostenosis and brachycephaly[16]. CYP26B1 mutations in both families led to a sig-nificantly attenuated ability to metabolize exogenouslyapplied retinoic acid, confirming the impact of dys-function of this gene on RA metabolism. Previousstudies have shown that CYP26B1 is responsible forATRA (all-trans retinoic acid) clearance in several tis-sues [17]. For example in T-cells, CYP26B1 is the onlyCYP26 enzyme up-regulated by ATRA and its expressionregulates retinoic acid dependent signals in T-cells [18].In addition, in many human cell lines CYP26B1 mRNA isinducible by retinoic acid treatment while in rodentscyp26b1 expression correlates with dietary vitamin Aintake [19-22].EXOC6B (exocyst complex component 6B) encodes aprotein homologous to Sec15 in Saccharomyces cervisiae.It belongs to a multiprotein complex (exocyst) requiredfor targeted secretion (exocytosis) which is crucial for cellpolarity, growth and communication [23]. In Drosophila,sec15 promotes Notch signaling, through specific vesicletrafficking of delta ligand, and has a role in asymmetricdivision of sensory precursors and neuronal fate deter-mination [24]. Drosophila neurons with sec15 mutationsshow loss of synaptic specificity and mislocalization ofproteins known to affect synaptic specificity in photore-ceptors [25]. Recently, Guichard et al. showed reducedtranscription of Notch signaling effectors HES1 and RBPJin knockdown Drosophila sec15 and in a human brainmicroendothelial cell line with abnormal EXOC6B func-tion [26]. Notch signaling plays a pivotal role in theregulation of many fundamental cellular processes, suchas acquisition of specific fates in context-dependentmanner, differentiation and lineage decisions duringembryonic development, neurogenesis, as well as mor-phogenesis involving regulation of left-right asymmetry[27,28]. Perturbations of the Notch pathway have beenreported in human developmental disorders whichdemonstrate a variety of symptoms, including ID and/or skeletal abnormalities (e.g. Allagile syndrome andspondylocostal dysostosis [29,30]).We report the first description of the clinical andfunctional consequences of hemizygous deletion of thetwo genes, CYP26B1 and EXOC6B, located in chromo-some region 2p13, and which are respectively, involvedin retinoic acid and Notch signaling pathways of criticalimportance to normal human fetal development.Materials and methodsWhole genome array CGH analysisGenomic DNA was extracted from peripheral blood usingthe PUREGENE DNA Isolation Kits (Gentra, Minneapolis,MN) for Subject 1 and Citogene® DNA isolation kit(Citomed, Portugal) for Subject 2. For Subject 1, Agilent105 K array (version 4.0, June 2006, Agilent Technolo-gies, CA, USA) analysis was performed as previously de-scribed.25 CNV selection was done by Agilent DNAAnalytics (version 3.5.14, Agilent Technologies) using theADM-2 algorithm (cutoff 6.0), followed by a filter to se-lect regions with three or more adjacent probes and aminimum average log2 ratio + 0.25 [31]. The deletion ofSubject 1 was de novo, as determined by FISH usingprobe RP11-91 F23.For Subject 2, the aCGH analysis was performed usinga CGH Agilent 180 K custom array design accessiblethrough the gene expression omnibus (GEO) accessionnumber GPL15397. Previously published protocol wasWen et al. Orphanet Journal of Rare Diseases 2013, 8:100 Page 2 of 12 [32]. Image analysis was performed using theacross-array methodology described previously [33].CGH data was analyzed using Nexus Copy Number 5.0software with FASST Segmentation algorithm. The dele-tion was determined to be de novo using the same arrayfor both parents.For both cases, the array design, database consultationand comparative analysis was performed using genomebuild 36.1/HG18.Functional studiesImmortalized EBV transformed lymphoblastoid cell lines(LCL) from Subject 1 were cultured in RPMI mediacontaining 10% FBS, 50 units/mL penicillin, 50 μg/mLstreptomycin. Cells were maintained in a humidified 37°Cincubator with 5% CO2. Control cells were obtained fromhealthy subjects, the majority of which aged 28–50 years.RNA expressionTotal RNA was extracted using an RNeasy Plus Mini kit(Qiagen) from Subject 1-derived LCLs and whole bloodcollected in Tempus tubes. Aliquots (~500 ng) of thetotal RNA extracts prepared from were subsequentlyreverse-transcribed into cDNA using GeneAmp GoldRNA PCR Core Kit (Applied Biosystems).The expression of EXOC6B and CYP26B1 was firstassessed in RNA extracted from control whole bloodand control LCLs by real-time qPCR using the ABIPRISM 7300 Sequence Detection System (Perkin-ElmerApplied Biosystems). The specific nucleotide sequencesfor primers of EXOC6B and CYP26B1 were as follows:EXOC6B Forward 5’-GAC CTC ATT GCC TTT CTTCGT A-3’, Reverse 5’-CAA GCT GAC ATA CAC GCTGT-3’(mapped to exons 18–19); CYP26B1 primer set 1;Forward 5’-ACA CAG GGC AAG GAC TAC T-3’, Re-verse: 5’-GCA TAG GCC GCA AAG ATC A-3’(mappedto exon 4–5); CYP26B1 primer set 2: Forward: 5’-CTACCT GGA CTG CGT CAT CA-3’, Reverse: 5’-CCCGGA TGC TAT ACA TGA CA-3’ (mapped to exon 5–6).EXOC6B had a detectable transcript, while no transcriptwas detected with CYP26B1 primers for exons 4–5 andexons 5–6 in control whole blood, nor transformedlymphoblasts.Real-time qPCR was performed for EXOC6B and itsdownstream genes HES1 and RBPJ using SYBR® GreenPCR Master Mix (Applied Biological Materials Inc). Pri-mer sequences were as follows: HES1 Forward: 5’-GAGCAC AGA AAG TCA TCA AAG C-3’ (mapped toexons 1–3), Reverse: 5’-TTC CAG AAT GTC CGC CTTC-3’; RBPJ Forward: 5’-GGG ATA GGA AAT AGT GACCAA GA-3’, reverse: 5’-GTG CTT TCG CTT GTCTGA GT-3’ (mapped to exons 7–8).Quantification of expression level of EXOC6B, HES1and RBPJ was performed in comparison to actin. AllPCR reactions were performed in triplicate, with themean of 2-ΔΔCt values [34] being used to determinemRNA levels for Subject 1 in comparison to the meanexpression level for three controls. Significance was cal-culated using the Student’s t test (VassarState StatisticalComputation website). Values were considered statisti-cally significant with a p-value of <0.05.Protein expressionLCLs were washed three times in PBS and incubated in1 mL RIPA buffer (Thermo Scientific, USA), supplementedwith Halt Phosphatase Inhibitor Cocktail (Thermo Scien-tific, USA) for 15 minutes on ice. Cell lysates werecentrifuged at 10,000 × g for 15 minutes at 4°C, andthe supernatants were used for Western blotting. Celllysate protein concentrations were determined using aDC™ Protein Assay (BioRad laboratories, USA). Westernblots containing cell-lysate aliquots (~30 μg) were pre-pared and immunoblotted using a polyclonal antibodydirected against human RBPJ or a monoclonal anti-body against human HES1 (Abcam, Cambrige, MA, USA).Three antibodies against human EXOC6B were tested inthis study (Sec15B(C-14) (goat polycolonal antibody):sc-34375, Santa Cruz Biotechnology,Inc. Anti-EXOC6B anti-body (rabbit polycolonal antibody): ab116383, Abcam Inc.Anti-EXOC6B antibody (mouse polycolonal antibody):H00023233-B01P, Novus Biologicals) but did not providea clear or visible band at the expected size, amenable tointerpretation. To standardize the amounts of proteinloaded into each lane, the blots were reprobed with amonoclonal antibody directed against human β-actin(NovusBiologicals, Littleton, USA). The ECL WesternBlotting system was used to detect the amount of eachantibody bound to antigen and the resultant photographicfilms were analyzed by UV densitometry (GE HealthcareLife Sciences, Pittsburgh, USA). The absorbance valuesobtained for HES1 and RBPJ were then normalized relativeto the corresponding β-actin absorbance value. The aver-age of HES1 or RBPJ protein expression was obtained fromthree independent replicates from the Subject and the con-trol samples. P values for two tailed Student test were con-sidered statistically significant if <0.05.CYP26B1 expression upon ATRA inductionCells from seven control subjects (aged 2–42 years) andSubject 1 were plated in 6-well plates at a density of400,000 cells/mL in 3 mL and treated in triplicate with1 μM all-trans retinoic acid (ATRA) or an equal volumeof vehicle (ethanol) in triplicate and incubated in thedark for 72 hours. Following treatment, cells werepelleted and resuspended in 1 mL Tri-Reagent Solution(Life Technologies, Grand Island, NY) followed by re-peated pipetting to lyse the cells. RNA was then iso-lated according to manufacturers instructions and RNAWen et al. Orphanet Journal of Rare Diseases 2013, 8:100 Page 3 of 12 and quality were determined using a NanoDrop2000c spectrophotometer (Thermo Scientific, Waltham,MA) by measuring the absorbance at 260 nm and280 nm. Complimentary DNA (cDNA) was generatedfrom 1 μg total RNA by reverse transcription using theTaqMan reverse transcription reagents kit (Life Tech-nologies, Grand Island, NY), as previously described[17,35]. TaqMan Gene Expression Master Mix, PCRprimers, and fluorescent probes were obtained from Ap-plied Biosystems (Life Technologies, Grand Island, NY).Probes were labelled with the 5’ reporter dye FAM(CYP26A1 and CYP26B1) or VIC (GAPDH). Primerprobe pairs used were: CYP26A1 (Hs00219866_m1),CYP26B1 (Hs00175627_m1), and the endogenous con-trol GAPDH (RefSeq: NM_002046.3). Quantitative real-time PCR was conducted on a StepOnePlus Real-TimePCR instrument (Applied Biosystems, Foster City, CA)as previously described [17,35]. The fold-increase inCYP26A1 and CYP26B1 mRNA was calculated usingthe ΔΔCt method (fold difference = 2-ΔΔCt) by compar-ing the ATRA-treated cells to the vehicle treated con-trols. Differences between controls and patient weretested by t-test using GraphPad Prism 5.0. Values wereconsidered statistically significant with a p-value <0.05.Grubb’s test was used to determine if any of the sub-jects met the criteria of an outlier.The use of tissues was approved by the Committee forEthical Review of Research involving Human Subjects,University of British Columbia. Written informed con-sent was obtained from the patients’ parents for the pub-lication of this report and any accompanying images.ResultsClinical descriptionSubject 1This male was seen by a geneticist as a neonate becauseof dysmorphic features, including an asymmetric cryingface with a right facial nerve paralysis, a dysplastic right ear,brachycephaly (Figures 1 and 2) and mild contractures ofthe knees and elbows (which disappeared by five months).The pregnancy was complicated by maternal nausea andvomiting, with only a five pound maternal weight gain.Birth weight was on the 10th centile. A 2 mm secundumatrial septal defect closed in early infancy. His parents arenot consanguineous. A paternal great-uncle has intellectualdisability while another paternal great-uncle’s adult son hasab cdd eB. Subject 2A. Subject 1a b cFigure 1 Clinical presentation of Subject 1 and Subject 2. A. Subject 1 at age 2, 7, and 13 (a-c), demonstrating the facial appearance, withasymmetry, right facial nerve palsy, dysplastic and prominent ears. By age 13, the right ear was surgically corrected. B. Subject 2 demonstratinga) asymmetry of the jaw, b) left ear with thick helix, c) right ear (post surgery): small, dysplastic, cup-shaped, anteverted, and hypoplastic lobule,d) hands with slightly tapering fingers and e) normal body proportions except short neck.Wen et al. Orphanet Journal of Rare Diseases 2013, 8:100 Page 4 of 12“autistic like features” at age 40. The remainder of the fam-ily history is unremarkable.As an infant, he had difficulty transitioning to solidfoods, with gagging and vomiting. He walked at 13 months.At 22 months, he had only a few words. By three years,he was not using any words regularly although hewould use a word for a few days and then stop. Withmost activities, he was not able to stay on track, beingquite hyperactive and distractible. His clinical diagno-sis of autism was confirmed by an assessment usingADOS at age 2 ½ years. At 13 years of age, his academicfunctioning was somewhat variable, with mathematicsbeing comparable to the skill level of a ten year old, whilethe level for other subjects was comparable to an eightyear old.At 22 months, height was above the 50th centile, weightwas below the 10th centile, and OFC was on the 3rd cen-tile. He had brachycephaly, which had been seen in in-fancy. The metopic suture, which was open at 7 months,was closed at 22 months. The left ear was low set, whilethe right was dysplastic, prominent, with a thin helix andan abnormal crural fold. His face appeared to be asym-metric, due to the right facial nerve palsy. Eye examinationwas normal except for right eyelid paralysis, secondary tothe right facial nerve palsy. The remainder of the physicalexamination was unremarkable.Chromosome testing was normal, including FISH at22q11.2. A CT scan of the brain was normal. Skull radio-graphs at age 7 months showed a shortened anterior-posterior diameter relative to width. There was slightbacd eSubject 1 Subject  2Figure 2 Skull and skeletal abnormalities in two subjects. a, b) brachycephaly in Subject 1 (a) and 2 (b); c) occipital asymmetry in Subject 1;d) congenital C1-C2 vertebral block; e) accentuation of dorsal kiphosis (arrow) in subject 2.Wen et al. Orphanet Journal of Rare Diseases 2013, 8:100 Page 5 of 12 of the appearance of the orbits, with the left sidea little larger and slightly more prominent superiorlaterally(Figure 2). No obvious sutural stenosis was evident. Atage 14, a C-spine radiograph was normal.Subject 2The Subject is a 9 year old boy referred for a geneticsconsultation because of delayed milestones and dys-morphic faces. His parents are non-consanguineous andhe has one healthy sister. His maternal uncle died at theage of 3 months, of unexplained sudden death. No otherfamily history of developmental problems or congenitalanomalies was reported. At the age of 3 years, he hadtwo episodes that raised the suspicion of absence sei-zures, but the EEG was normal. Delayed developmentwas noted, especially in language. He spoke few wordswith no sentences. Mild motor delay also was described,with unaided walking starting at 16 months.At four years, his growth parameters were normal(weight: 75th percentile; height: 50-75th percentile andOFC: 50-75th percentile). At five years he had a globaldevelopmental quotient of 53.4 on the Griffiths MentalDevelopmental Scale evaluation. His I.Q. at the age of7 years based on the Wechsler Intelligence Scale was 73(borderline mental retardation), with a verbal scale of 66and performance scale of 89. The proband was dysmorphicwith a triangular face, brachycephaly, hypertelorism, up-slanting palpebral fissures, thin lips, hypertrophic gums, apointed chin and short neck (Figure 1). He had abnormalears (asymmetric, dysplastic and low-set). The right earwas small, cup-shaped, anteverted and the lobule was hy-poplastic. The left ear was bigger than the right, with athick helix. Asymmetry of the jaw was noted, with the leftside longer than the right side. His fingers were slightly ta-pering. Radiographs revealed congenital C1-C2 vertebralfusion, with accentuation of dorsal kyphosis (Figure 2).Subject 2 displayed stereotypies, aggressive behavior,hyperactivity (including jumping if agitated) and atten-tion deficit. Brain MRI, echocardiogram, abdominalultrasound, ophthalmology and ENT examinations werenormal. Radiographs of upper and lower limbs, pelvisand rib cage did not reveal abnormalities. Chromosome,FISH for the 22q 11.2 region, molecular testing for Fra-gile X and metabolic studies (plasma aminoacids, urineorganic acids, CDT, creatine and guanidinoacetic acid inurine, 7-dehidrocholesterol, lactate, pyruvate and ammo-nia) were normal.The clinical description of the two subjects is summa-rized in Additional file 1: Table S1.Array CGHFor Subject 1, aCGH revealed a 0.78 Mb de novo dele-tion at chromosome region 2p13.2-13.3 (72,140,702-72,924,626), containing 2 genes. Subject 2 had a 4 Mbde novo deletion detected at chromosome region 2p13.1-p13.3 (chr2:70,748,414-74,840,026), containing 62 genes(Figure 3A and 3B). The region of overlap between bothcases is 0.78 Mb, located between the 72,140,702-72,924,626 genomic positions and encompasses theCYP26B1 and EXOC6B genes.Functional studies of EXOC6B and notch effector genesHES1 and RBPJ in subject 1Significant reduction of RNA expression for all threegenes (EXOC6B, RBPJ and HES1) was detected in lym-phoblasts from Subject 1 in comparison to three con-trols (Figure 4A). Protein expression for both HES1 andRBPJ was also significantly reduced in his lymphoblasts(Figure 4B and 4C), however, EXOC6B protein levelcould not be assessed due to a number of non-specificbands or a single band of an unexpected size in controlsobtained with three commercial EXOC6 antibodies.We noted that in control lymphoblasts, the baselinelevel of HES1 and RBPJ expression was significantly higherthan EXOC6, possibly due to the fact that EBV viral pro-tein EBNA2 induces RBPJ expression [36] (Figure 4A). Toeliminate the effect of EBV transformation on RBPJ andHES expression, whole blood was used from Subject 1and the controls. Similarly to the lymphoblasts, RNA ex-pression of EXOC6 and RBPJ was significantly reduced inthe Subject’s whole blood in comparison to a control.However, HES1 expression was significantly higher inwhole blood of Subject 1 than in the control bloods(Figure 5). Protein from whole blood was not availablefor assessment of RBPJ and HES1 expression levels.ATRA induction of CYP26B1 expressionEBV transformed lymphoblasts from seven controls andSubject 1 were treated with 1 μM ATRA for 3 days. Toobtain a quantitative comparison of the ATRA inductionof CYP26B1 in Subject 1 LCLs versus LCLs from con-trols, the fold difference in CYP26B1 mRNA inductionwith ATRA treatment was calculated for Subject 1 andcontrols in comparison to the vehicle treated cells.ATRA induction of CYP26B1 mRNA in LCLs from onecontrol subject (56.7 ± 3.7 fold) was determined to be anoutlier by Grubb’s test from the other controls and wasexcluded from further analysis. The CYP26B1 mRNAfold-induction from the six remaining control cells wasaveraged to show the range in induction in control LCLs(Figure 6A). CYP26B1 mRNA induction with ATRAtreatment was significantly less in Subject 1 compared toaveraged control cells (1.9 ± 1.5 fold in subject and 12.9 ±7.3 fold in controls,) (Figure 6A).The relatively minimal expression of CYP26B1 inATRA-treated cells from Subject 1 raised the possibilityof compensatory expression of CYP26A1 to regulate ret-inoic acid levels. ATRA-treatment resulted in minimalWen et al. Orphanet Journal of Rare Diseases 2013, 8:100 Page 6 of 12 of CYP26A1 mRNA in all LCLs which is inagreement with the lack of CYP26A1 induction andoverall low to undetectable expression in blood [18].CYP26A1 induction was not significantly different incontrol LCLs (3.4 ± 3.4 fold) compared to Subject 1LCLs (0.9 ± 0.9 fold) as shown in Figure 6B. Thus,CYP26A1 was not induced to a greater extent in re-sponse to ATRA treatment in cells with a deletion ofCYP26B1. To exclude the possibility that CYP26A1 ex-pression was higher in Subject 1 cells compared to con-trol cells prior to ATRA treatment, we compared theΔCt values of the vehicle-treated cells and found no differ-ences (P > 0.05). Hence, it does not appear that CYP26A1mRNA expression is upregulated to compensate for re-duced CYP26B1 expression in Subject 1 LCLs. Whileupregulation of Cyp26a1 has been observed in Cyp26b1−/−mice at specific developmental stages and tissues duringmouse organogenesis, overall, the expression patterns andfunction of Cyp26a1 and Cyp26b1 are considered non-overlapping during mouse development [37-39].DiscussionWe report a 2p13.1-p13.3 microdeletion observed in twosubjects with clinical effects on the cognitive function (IDand language delay), behaviour (hyperactivity), and devel-opment of the craniofacies (facial asymmetry, unusuallyshaped and asymmetric ears and brachycephaly). Skulland vertebral bone abnormalities included slight asym-metry of the appearance of the orbits and delayed closureof the metopic suture in Subject 1 and congenital C1-C2vertebral fusion, and accentuation of dorsal kyphosis inSubject 2. Previous cases with cytogenetic deletions/Subject 2Subject 1Subject 1Subject 2BAFigure 3 Array CGH result for Subject 1 and 2. A. Whole genome array profiles in Subject 1 and 2 showing microdeletions of 2p13; B. Genecontent for the microdeletions in Subject 1 and 2.Wen et al. Orphanet Journal of Rare Diseases 2013, 8:100 Page 7 of 12 of this region all had developmental delayand the majority had head/facial anomalies, ear andskeletal abnormalities [1,2].The overlapping deleted region of our two cases encom-passes two genes, EXOC6B and CYP26B1, both of whichshowed altered function in whole blood and/or LCLs fromSubject 1. The reduced expression of EXOC6B in LCLsand whole blood could be the cause of the observedperturbed Notch signaling (i.e. HES1 and RBPJ expressionchange), considering the similar effect of EXOC6B knock-out on Notch signaling in Drosophila.20 The reduction ofRNA expression of EXOC6B and RBPJ was concordantbetween LCLs and whole blood, and the expression of theRBPJ protein also was reduced in Subject 1 lymphoblasts.However, HES1 had reduced RNA and protein expressionin LCLs and increased RNA expression in whole blood.The discrepancy in the pattern of abnormal expression ofHES1 in different cell types could be due to the differencein the Notch signaling pathway in lymphoblasts whichcontain dedifferentiated B cells vs. whole blood whichcontains multiple differentiated cell lineages. Cell-specificover or underexpression of Hes1 has been reported in dif-ferent regions of the brain in Rbpj knockout mice [40].EXOC6B germline mutations or deletions have yet notbeen reported in humans. A homozygous mutation inEXOC6B’s paralogue EXOC6A has been reported in micewith hemoglobin-deficit (hbd) due to defective iron trans-port in the endocytosis cycle [41] while haploinsufficiencyof EXOC6A due to a 0.3 Mb microdeletion at 10q23.33,was reported in a family with nonsyndromic bi- andunilateral optic nerve aplasia [42]. Interestingly, thismicrodeletion also included two CYP genes, CYP26A1RBPJ 50KD42KDS1               C1           C2            C3-actinB CRelative  Protein levels*S1             C1             C4 C5*S1              C1            C2 C3Relative  Protein levelsHES1-actin30KD42KDS1             C1            C4           C5AEXOC6B                                   RBPJ                                           HES1*****Figure 4 mRNA and protein expression in Subject 1 and control lymphoblast cells. A. Scatter graph of mRNA expression of EXOC6, RPJBand HES1in lymphoblasts. Values for EXOC6, RPJB and HES1 mRNA expression were normalized to the corresponding ß-actin mRNA levels. Theresults derived from at least three sets of samples and the mean level of each sample is represented in the graph, horizontal bar is an averagevalue between three controls. The difference between the study sample and the average of the controls has been evaluated by student t-test(*, p < 0.05; **, P < 0.01). B. RBPJ protein expression. C. HES1 protein expression. C1-C5 indicate control samples from the lymphoblasts, S1indicate study sample from Subject1. Cell lysates were analyzed by SDS-PAGE and immunoblotting with membrane probed for RBPJ, HES1and ß-actin, Values for RBPJ and HES1 expression were normalized to the corresponding ß-actin levels. The results derived from at leastthree sets of samples and the mean value of each sample is represented in the bar graph. The difference between Subject1 and theaverage of three controls has been evaluated by student t-test (*, p < 0.05).Wen et al. Orphanet Journal of Rare Diseases 2013, 8:100 Page 8 of 12 CYP26C1. In contrast to very little information ongenetic defects of EXOC6B and their role in disease,genetic abnormalities of HES1 and RBPJ have been as-sociated with a number of developmental defects invertebrates. Homozygous Hes1 and Rbpj knockoutmice showed severe developmental defects and lethal-ity [43,44], while Rbpj heterozygous knockout micedemonstrated learning deficits [29]. In humans, in-creased expression of HES1 was reported in Down syn-drome [45] and recent exome sequencing studiesrevealed heterozygous mutations in RBPJ and reducedexpression of HES1 in two families with Adams-Oliversyndrome, associated with congenital cutis aplasia, ter-minal limb abnormalities (asymmetric shortening ofthe hands and feet in one of the families) and a rangeof cognitive functioning (from intellectual disability tonormal) [46].The deletion of CYP26B1 gene in both our Subjects isalso likely to contribute to their abnormal phenotype, basedon abnormal RA metabolism in Subject 1, as evidenced bysignificantly attenuated induction of CYP26B1 expressionwith ATRA in comparison to controls. To the best of ourknowledge, there are no reports on the effect of CYP26B1gene haploinsufficiency in humans. Previously, in two otherfamilies, two different homozygous mutations of CYP26B1have been reported, resulting in lethality, skeletal and cra-niofacial abnormalities, including fusion of long bones,calvarial bone hypopasia and craniosynostosis [16]. In one00.511.522.53EXOC6B patientEXOC6B controlRBPJ patientRBPJ controlHES1 patientHES1 controlEXOC6B                                 RBPJ                                         HES1 *  *  ** Figure 5 mRNA expression of EXOC6, RPJB and HES1 in whole blood of Subject 1 and controls. Values for EXOC6, RPJB and HES1 mRNAexpression in whole blood were normalized to the corresponding ß-actin mRNA levels. The results derived from at least three sets of samplesand the mean level of each sample is represented in the scatter graph, horizontal bar is an average value between three controls. The differencebetween subject 1 and the average of the controls has been evaluated by student t-test (*, p < 0.05; **, P < 0.01).AP < 0.05n.s.BFigure 6 Induction of CYP26B1 and CYP26A1. A. Fold induction of CYP26B1 mRNA expression. CYP26B1 was induced significantly more (*P <0.05) in the control cells as compared to the Subject 1 (S1) cells with a chromosomal deletion of CYP26B1 after treatment with ATRA. B. Inductionof CYP26A1 mRNA expression in ATRA-treated cells. There was no significant difference (n.s.; P > 0.05) in CYP26A1 induction between the controlcells and Subject 1 cells.Wen et al. Orphanet Journal of Rare Diseases 2013, 8:100 Page 9 of 12 the families with the hypomorphic mutation, brachyceph-aly and wide sagittal sutures were noted. The two mutation-bearing constructs had attenuated ability to metabolizeATRA (36% and 86%) [16]. In our Subjects, the decreasedRA catabolism and increased sensitivity to RA, as a conse-quence of CYP26B1 deletion could explain features similarto those noted by Laue et al. [16] (e.g. brachycephaly forboth subjects, and for Subject 1, the delayed closure of themetopic suture) and in general compromise the craniofacial,skeletal development and neuronal functioning. With regardto the later phenotype, it is of interest that Subject 1 had anasymmetric crying face as a consequence of a right facialnerve palsy which previously was associated with RA expos-ure or early embryonic insult [11,47]. The phenotype of thesubjects is in agreement with developmental effects ofCYP26B1 deletion in experimental animals [37]. CYP26B1null mice have craniofacial abnormalities, exhibit abnormalear development and other bone and cartilage deformities[13]. Interestingly, the truncated limbs observed inCYP26B1−/− mice were absent in the patients. Similarly, inthe zebrafish CYP26B1 deletion has been shown to re-sult in overall defective craniofacial cartilage develop-ment with smaller head, severely decreased number ofvagal branchiomotor neurons and defective or absentjaw cartilage [48].It is intriguing to note the lethal phenotype in twosubjects reported by Laue et al. [16] due to homozygousmutations of CYP26B1 in comparison to the survivaland developmental abnormalities in our subjects withhemizygous CYP26B1 deletion. The presence of onenormal copy of the gene in each of our subjects and theefficiencies of the other two remaining RA catabolizingCYP26 genes (CYP26A1 and CYP26C1), possible com-pensatory changes in other proteins known to regulateretinoic acid, such as RALDH [8,49,50] and environmen-tal influences, such as diet and pharmacological treat-ment, [6,51] all could have an effect on the phenotypes.Phenotypic variability also was noted for carriers ofCYP26A1, CYP26C1 and EXOC6A deletions within onefamily [42], ranging from normal vision, to uni- and bi-lateral optic nerve hypoplasia and variable levels of cog-nitive functioning (normal to impaired).The combined effect of deletion of both EXOC6B andCYP26B1 on Notch and RA signaling, and consequentlythe phenotype, also should be considered in our sub-jects. Interaction of RA and Notch signaling in deter-mination of left/right asymmetry and segmentation hasbeen reported by Echeverri and Oates [52] who notedthe requirement of Rbpj function for expression of RAcatabolizing enzyme Cyp26a1 which in turn, is neededfor left/right symmetric cyclic gene expression. Vermotet al. [53] demonstrated that reduced levels of RA wereassociated with abnormal Hes1 expression and asym-metry in mouse embryos, while Castella et al. [54]showed that addition of RA raises the level of HES1 pro-tein expression in in vitro cell culture.Subject 2 had additional phenotypic features not notedin Subject 1, which might be explained by the larger sizeof the 2p13.1-13.3 deletion, interaction of its integralgenes and genetic background. For example, seizure-likeepisodes were not noted in Subject 1, but were presentin Subject 2 and a Decipher subject, # 257412, with de-velopmental delay, whose deletion of 2p12-13.3 was6.8 Mb (70,889,254-77,746,500), and his features in-cluded coarse faces, a flat malar region, myopathic hypo-tonia and prominent ears. The cervical fusion and spineabnormality are unique for Subject 2 and are of interest,considering the report of a subject with Klippel-Fielanomaly, who had a balanced inversion involvingchromosome 2p13 and congenital fusion of the cervicalspine, impairment of hearing, psychomotor retardation,speech limitation, short stature, spinal asymmetry andscoliosis [3]. The genes disrupted/deleted by this chromo-some rearrangement are unknown, however, it was specu-lated that CYP26B1 was involved based on the similarvertebral phenotype observed in zebrafish with Cyp26b1mutations [4].Our report is unique as it provides a new insight intothe phenotypic and functional consequences of hemizy-gous deletion of two genes implicated in Notch and Ret-inoic acid signaling. It also supports the previous cellline and animal model based observation of exocyst rolein Notch signaling. Further studies of exocyst complexfunction in patient cells would be of interest for under-standing of its role in human disease.Additional fileAdditional file 1: Table S1. Clinical features of Subject 1 and 2.Competing interestsThe authors have no competing interests to declare.Authors’ contributionJW, ERS, CN, NI, FL and PM: Conception, design, analysis and interpretationof data. Drafting the article or revising it critically for important intellectualcontent. SAF, GS, SL, CB, YQ, BY and SM: Analysis and interpretation of data.Drafting the article or revising it critically for important intellectual content.All authors read and approved the final manuscript.Authors’ informationPatricia Maciel and Evica Rajcan-Separovic are co-corresponding authors.AcknowledgmentsWe would like to sincerely acknowledge the families of the presented casesfor participation in the genetic studies and for allowing this publication.This work was supported by funding from the Canadian Institutes for HealthResearch (CIHR) (RT-64217; PI: MESL and MOP 74502; PI: ERS), Autism Speaks(PI: MESL) and Fundação para a Ciência e Tecnologia (PIC/IC/83026/2007.MES and ERS are Career Investigators with the Michael Smith Foundation forHealth Research. The experiments were supported in part by a US Nationalinstitutes of Health grant R01 GM081569 (NI and CN).Wen et al. Orphanet Journal of Rare Diseases 2013, 8:100 Page 10 of 12 study makes use of data generated by the DECIPHER Consortium. A fulllist of centres which contributed to the generation of the data is availablefrom and via email from for the project was provided by the Wellcome Trust.Author details1Child and Family Research Institute, Department of Pathology, University ofBritish Columbia, Vancouver, BC, Canada. 2Life and Health Sciences ResearchInstitute (ICVS), School of Health Sciences, University of Minho, Braga,Portugal. 3ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães,Portugal. 4Center for Medical Genetics Dr. Jacinto Magalhães, National HealthInstitute Dr Ricardo Jorge, Porto, Portugal. 5Genetics, Trillium Health Partners,Credit Valley Hospital Site, Mississauga, ON, Canada. 6Department ofPharmaceutics, School of Pharmacy, University of Washington, Seattle, WA,USA. 7Department of Pathology, VU University Medical Center, Amsterdam,The Netherlands. 8Child and family Research Institute, Department of MedicalGenetics, University of British Columbia, Vancouver, BC, Canada.Received: 21 February 2013 Accepted: 3 July 2013Published: 10 July 2013References1. 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Mol Cell Biol 2000, 20:6170–6183.doi:10.1186/1750-1172-8-100Cite this article as: Wen et al.: Phenotypic and functional consequencesof haploinsufficiency of genes from exocyst and retinoic acid pathwaydue to a recurrent microdeletion of 2p13.2. Orphanet Journal of RareDiseases 2013 8:100. Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at et al. Orphanet Journal of Rare Diseases 2013, 8:100 Page 12 of 12


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