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Understanding the impact of 1q21.1 copy number variant Harvard, Chansonette; Strong, Emma; Mercier, Eloi; Colnaghi, Rita; Alcantara, Diana; Chow, Eva; Martell, Sally; Tyson, Christine; Hrynchak, Monica; McGillivray, Barbara; Hamilton, Sara; Marles, Sandra; Mhanni, Aziz; Dawson, Angelika J; Pavlidis, Paul; Qiao, Ying; Holden, Jeanette J; Lewis, Suzanne M; O’Driscoll, Mark; Rajcan-Separovic, Evica Aug 8, 2011

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RESEARCH Open AccessUnderstanding the impact of 1q21.1 copynumber variantChansonette Harvard1,2†, Emma Strong1,2†, Eloi Mercier3, Rita Colnaghi4, Diana Alcantara4, Eva Chow5,Sally Martell1,2, Christine Tyson6, Monica Hrynchak6, Barbara McGillivray7, Sara Hamilton7, Sandra Marles8,Aziz Mhanni8, Angelika J Dawson8, Paul Pavlidis3, Ying Qiao1,2,7, Jeanette J Holden9,10,11, Suzanne ME Lewis1,7,10,Mark O’Driscoll4* and Evica Rajcan-Separovic1,2*AbstractBackground: 1q21.1 Copy Number Variant (CNV) is associated with a highly variable phenotype ranging fromcongenital anomalies, learning deficits/intellectual disability (ID), to a normal phenotype. Hence, the clinicalsignificance of this CNV can be difficult to evaluate. Here we described the consequences of the 1q21.1 CNV ongenome-wide gene expression and function of selected candidate genes within 1q21.1 using cell lines fromclinically well described subjects.Methods and Results: Eight subjects from 3 families were included in the study: six with a 1q21.1 deletion andtwo with a 1q21.1 duplication. High resolution Affymetrix 2.7M array was used to refine the 1q21.1 CNVbreakpoints and exclude the presence of secondary CNVs of pathogenic relevance. Whole genome expressionprofiling, studied in lymphoblast cell lines (LBCs) from 5 subjects, showed enrichment of genes from 1q21.1 in thetop 100 genes ranked based on correlation of expression with 1q21.1 copy number. The function of two topgenes from 1q21.1, CHD1L/ALC1 and PRKAB2, was studied in detail in LBCs from a deletion and a duplicationcarrier. CHD1L/ALC1 is an enzyme with a role in chromatin modification and DNA damage response while PRKAB2is a member of the AMP kinase complex, which senses and maintains systemic and cellular energy balance. Theprotein levels for CHD1L/ALC1 and PRKAB2 were changed in concordance with their copy number in both LBCs. Adefect in chromatin remodeling was documented based on impaired decatenation (chromatid untangling)checkpoint (DCC) in both LBCs. This defect, reproduced by CHD1L/ALC1 siRNA, identifies a new role of CHD1L/ALC1in DCC. Both LBCs also showed elevated levels of micronuclei following treatment with a Topoisomerase IIinhibitor suggesting increased DNA breaks. AMP kinase function, specifically in the deletion containing LBCs, wasattenuated.Conclusion: Our studies are unique as they show for the first time that the 1q21.1 CNV not only causes changesin the expression of its key integral genes, associated with changes at the protein level, but also results in changesin their known function, in the case of AMPK, and newly identified function such as DCC activation in the case ofCHD1L/ALC1. Our results support the use of patient lymphoblasts for dissecting the functional sequelae of genesintegral to CNVs in carrier cell lines, ultimately enhancing understanding of biological processes which maycontribute to the clinical phenotype.* Correspondence: m.o-driscoll@sussex.ac.uk; eseparovic@cw.bc.ca† Contributed equally1Child and Family Research Institute, Molecular Cytogenetics and ArrayLaboratory, 950 West 28th Avenue, Vancouver, BC, Canada4Human DNA Damage Response Disorders Group, Genome Damage &Stability Centre, University of Sussex, Brighton, UKFull list of author information is available at the end of the articleHarvard et al. Orphanet Journal of Rare Diseases 2011, 6:54http://www.ojrd.com/content/6/1/54© 2011 Harvard et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.BackgroundCopy number changes of 1q21.1 chromosomal region(OMIM 612474 and 612475) have been associated withvariable phenotypes which include ID and/or autism[1,2], schizophrenia [3-5], congenital heart anomalies[2,6-8], dysmorphic features [1,6,7] or a normal pheno-type [1,2]. Deletions and duplications of 1q21.1 weredetected in 0.24% and 0.12% of cases respectively [9], andin 1/4737 controls [2]. The 1q21.1 critical region spansapproximately 1.35 Mb (from 145 to 146.35 Mb, accord-ing to NCBI build 36) [2] and includes at least 12 genes.The cause of the phenotypic variability associated with1q21.1 copy number variant (CNV) remains largely unex-plained; however recent studies show that the presence of“two hit” CNVs can contribute to variability associatedwith CNVs that escape syndromic classification [10].The impact of the 1q21.1 CNV, beyond the clinicaldescription of affected subjects, is unknown. Tradition-ally, the functional impact of CNVs is studied in mousemodels where expression changes in 83% of genes fromCNVs were reported in at least one, but frequently inseveral, mouse tissues studied [11,12]. Mouse models ofhuman microdeletion/microduplication disorders suchas DiGeorge [13] and Smith Magenis syndrome [14],also helped to detect expression changes at the mRNAand protein levels of genes integral to CNVs and iden-tify the critical candidate genes for the phenotype (e.g.transcription factors Tbx1 for DiGeorge and RAI1 forSmith Magenis syndrome). Subsequent studies ofmutant forms of these genes in transfected human celllines showed their abnormal function at the cellularlevel (i.e. changed transcriptional activity and/or abnor-mal sub-cellular localization/stability of the protein[15,16]). Unfortunately, functional consequences ofgenes integral to CNVs in cells/tissues from carriers arerarely studied, due to unavailability of appropriatehuman tissues and the rarity of patients with individualCNVs [17]. Nevertheless, in rare cases where humanlymphoblasts were used to assess gene expression inCNV carriers, changes within the CNV and genomewide were noted [18,19] suggesting that peripheralblood cells can be used for assessment of the effect ofgene copy number change. Subsequent studies of thefunction of genes showing expression changes in cellsfrom CNV carriers have not yet been reported.Our study aimed to understand the impact of the1q21.1 CNV on gene expression genome wide as well ason the function of a selection of its integral genes in lym-phoblasts cell lines from clinically well described subjects.MethodsSubjectsEight subjects were included in the study and their clini-cal description provided in Additional File 1, Table S1.They belong to three families (family A, B and C with 3,3 and 2 subjects, respectively). Individuals A1, A2, A3,C1, and C2 were enrolled in a research based arrayCGH screening for pathogenic CNVs. The detailed cri-teria for enrollment were described in Qiao et al. (2010)[20]. The array CGH study was approved by the Univer-sity of British Columbia Clinical Research Ethics Board.Subjects B1 and B2 were ascertained via a clinical genet-ics service. They had normal karyotypes and Fragile Xtesting. B1’s brother, B3, was also ascertained throughclinical genetic service because of the family history of1q21.2 CNV.Whole Genome ArraysThe 1q21.1 CNV was detected in all subjects usinginitial lower resolution whole genome array analysis aspreviously described [20]. Seven of eight subjects werealso analysed subsequently using the new AffymetrixCytogenetics Whole-Genome 2.7 M Array (DNA wasnot available from B2 for high resolution array analysis).This higher resolution array contains approximately400,000 SNP markers and 2.3 million non-polymorphicmarkers, with high density coverage across cytogeneti-cally significant regions. Data was collected using eitherGeneChip® Scanner 3000 7 G or GeneChip® Scanner3000 Dx and CEL files were analyzed using AffymetrixChromosome Analysis Suite software (ChAS v.1.1). Theannotation file used in our analysis can be found on theAffymetrix website, listed as ArrayNA30.2 (hg18). Addi-tional CNVs detected with the high resolution arraywere compared with the Database of Genomic Variantshttp://projects.tcag.ca/variation for overlap with copynumber variants in controls using previously describedcriteria for defining common variants [20].Fluorescent in-situ hybridization (FISH)Rearrangements at 1q21.1 were confirmed by FISH fol-lowing previously described protocols [21]. FISH probesused are listed in Additional File 1, Table S1.Whole genome expressionRNA from EBV (Epstein Barr Virus) transformed lym-phoblastoid cell lines was used to study gene expressionin subjects with a 1q21.1 microdeletion (A1-3), micro-duplication (C1 & C2), and in 3 normal controls. Tran-script levels were assayed using a commercial wholegenome expression array (Illumina, HumanRef-8 v3.0Expression BeadChip) using standard protocols. Arrayhybridization, washing, blocking, and streptavadin-Cy3staining were also done according to standard protocols(Illumina). The BeadChip was then scanned using anIllumina BeadArray Reader to quantitatively detectfluorescence emission by Cy3. Eight arrays were run inparallel on a single BeadChip. Each array contained ~Harvard et al. Orphanet Journal of Rare Diseases 2011, 6:54http://www.ojrd.com/content/6/1/54Page 2 of 1224,500 well-annotated transcripts (NCBI RefSeq data-base Build 36.2, Release 22), present multiple times on asingle array.Expression Data AnalysisBackground-corrected intensity values were generatedfor each probe using GenomeStudio software (Illumina).Subsequent analyses were carried out in R http://www.R-project.org/. The data were quantile normalized anddifferential expression with respect to 1q21.1 copy num-ber analyzed using limma [22], with Benjamini-Hoch-berg multiple test correction to control the falsediscovery rate (FDR). This yields a ranking of the genesused in subsequent analyses.The ranking of genes from the 2.5 Mb and 5 Mbflanking regions of 1q21.1 (57 and 150 genes respec-tively) were examined in the full ranking provided bythe analysis described above, and tested for enrichmentusing the Wilcoxon rank-sum test as well as the hyper-geometric distribution considering just the 100 geneswith the highest expression/1q21.1 copy numbercorrelation.In silico functional analysis of top 100 genesGenes which ranked highest (top 100 genes) in theexpression/1q21.1 copy number correlation analysiswere selected for further in silico functional analysis. Anover-representation analysis (ORA) for Gene Ontology(GO) terms was performed using ermineJ http://www.chibi.ubc.ca/ermineJ/[23]. GO terms considered includedbiological processes, molecular functions, and cellularcomponents. The ORA analysis was run using the fol-lowing settings: gene set sizes were restricted from to 3-200 genes and best scoring replicates were used for anyreplicate genes in the datasets.Functional studiesCell cultureEBV-transformed patient-derived LBCs were cultured inRPMI with 15% FCS (fetal calf serum), L-Gln and anti-biotics (Pen-Strep) at 5% CO2. The Werner syndromeLBCs (WRN) were from a WRN syndrome patienthomozygous for the p.Arg368X pathogenic mutation.A549 adenocarcinoma cells were maintained in MEMwith 10% FCS.Antibodies and Western blotting analysisAnti-CHD1L (CHDL1 21703a), MCM2, phospho-S10-histone H3 and b-tubulin were from Santa Cruz. Anti-bodies against AMPKb1, AMPKb2 (4148), AMPKa andAMPKa-pT172, ACC, ACC-pS79 and RAPTOR-pS792were obtained from Cell Signalling. Whole cell extractswere prepared by lysing cells in urea buffer (9 M urea,50 mM Tris-HCl at pH 7.5 and 10 mM 2-mercaptoetha-nol), followed by 15 s sonication at 30% amplitude usinga micro-tip (SIGMA-Aldrich). The supernatant wasquantified by Bradford Assay. For CHD1L and AMPK-b2 expression, differing amounts of whole cell extractswere separated by SDS-PAGE and Western blotting sig-nals were obtained following ECL (Pierce)-development.Densiometric quantification of scanned films wasachieved using the Image J Software.ATM- and ATR-dependent G2-MG2-M cell cycle checkpoint analysis was carried out aspreviously described [17]. Briefly, following irradiation(3 Gy IR for ATM-dependent or 7 J/m2 UV for ATR-dependent) cells were incubated for 4 h in the presenceof 200 ng/mL of Demecolcine prior to swelling, fixation(Carnoy’s) and staining as described below.Decatenation Checkpoint Assay (DCC)Exponentially growing LBCs were treated with 1 μMICRF193 (Meso-4,4’-(3,2-butanediyl)-bis(2,6-piperazine-dione) and 200 ng/mL of Demecolcine and incubatedfor 4 h. Cells were harvested, washed 1× in PBS andswollen in 75 mM KCl for 10 min before fixing withPBS containing 3% paraformaldehide, 2% Sucrose for 10min. Following a PBS wash cells were cytospun on topolylysine coated slides and treated with 0.2% triton X-100 for 1 min before staining with an anti-phospho-his-tone H3 polyclonal antibody and secondary detectionusing Cy3-conjugated anti-rabbit. Nuclei were counter-stained with 0.2 μg/mL 4,6-diamidino-2-phenylindoledilactate (DAPI) and viewed using Nikon E-400 micro-scope. Approximately 300 cells were counted pertreatment.CHD1L/ALC1 siRNA and ICRF193 treatmentCHD1L/ALC1 knock out in A549 epithelial lung cancercells was done using 20 nM Darmacon SmartPoolsiRNA oligos with Metafectine as the transfectionreagent according to the manufacturer’s instructions. 20h after addition of siRNA, cells were treated with 0.05μM ICRF193 and 200 ng/mL of Demecolcine and incu-bated for 4 h. For chromosome spreads cells were swol-len in 75 mM KCL (10 mins) and fixed in Carnoy’s(methanol:glacial acetic acid 3:1) for 10 mins, washed(PBS), dropped onto slides and air dried prior to stain-ing with Giemsa and imaged using a ZeissAxioplanmicroscope. Indirect immunofluoresence using anti-phospho-Ser10-Histone 10 was also carried out. At least100 mitotic spreads were counted per treatment.Pseudomitoses and Micronuclei determinationCells with entangled chromosomes were considered torepresent pseudomitoses. Their frequency was deter-mined relative to interphase cells (mean no. of inter-phase cells counted per treatment = 300).The levels of micronuclei (MN) were enumerated incytochalasin B-induced binucleate [24] cells followingexposure to and recovery from a low dose of ICRF193.The MN present in binucleate cells are derived from theHarvard et al. Orphanet Journal of Rare Diseases 2011, 6:54http://www.ojrd.com/content/6/1/54Page 3 of 12previous cell cycle. Exponentially growing LBCs weretreated for 16 hrs with 0.1 μM ICRF193. The drug wasremoved, cells washed in PBS and treated with cytocha-lasin B (1.5 μg/ml) for a further 24 hrs. Cells were pel-leted, fixed in Carnoy’s, stained with DAPI and,cytospun onto poly-L-Lysine coated slides and viewedusing a Nikon E-400 microscope. At least 100 binucleatecells were enumerated per treatment.ResultsClinical and genomic findingsThe clinical and genomic findings for all eight 1q21.1CNV carriers are presented in Additional File 1, TableS1 and Figure 1. The clinical assessment included prena-tal history and prenatal/newborn complications weredocumented in 5/8 cases. In addition, detailed clinicalevaluation of 1q21.1 CNV carriers, both affected as wellas those initially considered to be normal, was per-formed. This resulted in recognition of learning pro-blems of various degrees in all studied subjects,although 2/6 subjects (A3 and C2) had very subtlelearning difficulties as A3 did not complete secondaryschool training and C2 admitted having to work veryhard to pass grades. Learning difficulties of variabledegree were therefore common to all subjects, whileother features varied, within and between families.In family A, the phenotypes of three 1q21.1 deletioncarriers showed different severity despite identical1q21.1 gene content and almost identical 1q21.1 break-points (Additional File 1, Table S1 and Figure 1) asdetermined by high resolution 2.7 M Affymetrix array.In family B, phenotypes also differed between indivi-duals, with individual B3 showing the least severe phe-notype despite having the largest genomic imbalancewhich included both a deletion and a duplication. Infamily C, the affected proband (C1) inherited the dupli-cation from his father, who was apparently normal butreported mild ADHD as child (not treated) and difficul-ties in passing grades in school.The core genes seen in all subjects with a 1q21.1 CNVare PRKAB2, PDIA3P, FMO5, CHD1L/ALC1, BCL9,ACP6, GJA5, GJA8, GPR89B, GPR89C, PDZK1P1, andChr1 (q21.1)Figure 1 Comparison of genomic overlap for 1q21.1 CNVs. CNV breakpoints were determined using Affymetrix 2.7 M whole genome arrayfor all subjects except B2 whose breakpoints were determined using a SignatureChip WG v1.1. Red bars indicate deletion of 1q21.1 region whileblue bars indicate a duplication. The previously reported minimal deletion region is shown in green. Genes seen in the majority of our cases(core genes) are highlighted in yellow.Harvard et al. Orphanet Journal of Rare Diseases 2011, 6:54http://www.ojrd.com/content/6/1/54Page 4 of 12NBPF11. There were no secondary CNVs that could beconsidered pathogenic and contributing to thephenotype.Whole genome expression analysisGene expression analysis was performed for 3 subjectswith microdeletion (A1-3, from family A) two subjectswith microduplication (C1 and C2 from family C) and 3controls. Ranking of genes was based on correlation ofexpression changes and 1q21.1 copy number. Significantenrichment of gene transcripts from the 1q21.1 CNV(6/11 with probes on Illumina array) was detectedwithin the top 100 genes in our 1q21.1 copy number/expression correlation analysis (Additional File 1, TableS2 and Figure 2). Transcripts from these genes(PRKAB2, CHD1L/ALC1, BCL9, ACP6, GPR89A, andPDIA3P) are ranked higher in our analysis than wouldbe expected by chance (p = 2.5 × 10-14) and are posi-tively correlated with 1q21.1 copy number with theexception of PDIA3P.CHD1L/ALC1, a gene within the 1q21.1 CNV, was atthe top of the correlation list, i.e. the correlation of itsexpression and copy number change was the least likelyto have occurred by chance (p = 2.42 × 10-5, though notsignificant after multiple test correction). The p valuesfor the correlation of expression and 1q21.1 copy num-ber for all probes across all chromosomes is shown inAdditional File 2, Figure S1. We did not find anyFigure 2 Correlation of expression and copy number for probes from chromosome 1 expressed as log10 of the p values (seeMethods). The probes from 1q21.1 region are in black.Harvard et al. Orphanet Journal of Rare Diseases 2011, 6:54http://www.ojrd.com/content/6/1/54Page 5 of 12evidence that the 1q21.1 CNV influenced expression ofgenes flanking the CNV (2.5 or 5 Mb windows; Wil-coxon rank-sum test and hypergeometric tests p > 0.2,see Methods). Gene Ontology enrichment analysis didnot reveal any GO terms with more genes from the top100 than would be expected by chance.Gene function analysisGene function analysis was performed using LBCs fromB1 and C1. B1 represented the 1q21.1 deletion (Del)and C1 represented the 1q21.1 duplication (Dup). Twogenes, CHD1L/ALC1 and PRKAB2, from 1q21.1 werestudied because they ranked highest in the expression/1q21.1 copy number correlation analysis (CHD1L/ALC1position 1 and PRKAB2 position 10) and because theyhave functions in relevant cellular processes (see belowand discussion for details). The protein expression ofthese genes was determined using Western blotting inpatient LBCs. Reduction of protein level for bothCHD1L/ALC1 and PRKAB2 was seen in the LBCs with1q21.1 Del and an increase in the LBCs with 1q21.1Dup in comparison to the control (Figure 3A and 3Band 5A respectively).Functional assays for CHD1L/ALC1CHD1L/ALC1 (hereafter referred to as CHD1L) has beenimplicated in chromatin remodeling and DNA relaxationprocess required for DNA replication, repair and tran-scription [25]. Both depletion and over-expression ofCHD1L have been implicated in impaired chromatinremodeling during DNA single strand break repair [26]suggesting that it is a dosage-sensitive gene with a role inDNA Damage Response (DDR). The DDR incorporatesDNA repair processes as well as signal transduction pro-cesses and is coordinated by two protein kinases ATM(Ataxia Telangiectasia Mutated) and ATR (Ataxia Telan-giectasia Mutated Rad3-related) that sense DNA damageand co-ordinate appropriate cell cycle checkpoint activa-tion, DNA repair and apoptosis [27].We set out to probe DDR function in 1q21.1 CNVLBCs by initially examining the ATM and ATR-depen-dent G2-M checkpoint via mitotic index enumerationfollowing ionising radiation (IR; for ATM-dependentarrest) or UV irradiation (for ATR-dependent arrest)respectively. LBCs with a deletion or duplication of1q21.1 exhibited normal arrest, as evidenced by anincrease in G2 cells and decrease in mitotic cell index,suggesting functional ATM and ATR-dependent check-point activation (data not shown). But, in the course ofthis analysis we noticed elevated levels of ‘pseudomitosis’in LBCs containing 1q21.1 Del and Dup containing celllines, which prompted more detailed analysis of theirfrequencies in the 1q21.1 Del and Dup cell lines. Pseu-domitotic cells exhibit catenated entangled chromatidsand their presence indicates sub-optimal DecatenationCheckpoint (DCC) activation (Figure 3C). The DCC is afunctional cell cycle checkpoint, involving proteins suchas ATR, ATM, WRN, MDC1, BRCA1 and RAD9, thatdelays cells in G2 phase until DNA is fully decatenated[28]. Chromosome catenation is a normal by-product ofDNA replication as replication forks attempt to mergeproducing intertwined catenated sister chromatids (Fig-ure 3C). Topoisomerase II alpha (Topo IIa) specificallyfunctions to decatenate/untangle these chromosomes bytransient introduction of a DNA double strand break(DSB) allowing one strand to pass through the otherthereby facilitating completion of DNA replication andfaithful chromosome segregation (Figure 3C). DCC canbe activated following treatment with a bisdioxopipera-zine Topo II catalytic inhibitor that prevents Topo-II-dependent DSB formation (e.g. ICRF193).Interestingly, we found that LBCs carrying a Del orDup of 1q21.1 failed to arrest in G2 following Topo IIinhibition, and instead, exhibited elevated pseudomitosissimilar to WRN-defective cells from a patient with Wer-ner syndrome (OMIM #277700, Figure 3D), which areknown to exhibit defective DCC activation [29]. Consid-ering that CHD1L functions as a chromatin remodeler[26], and that catenated chromosomes are a conceivableoutcome of an inability to efficiently manipulate chro-matin structure, we sought to determine whether reduc-tion of CHD1L specifically could underlie thisphenotype. Using careful titration of CHD1L siRNA inA549 cells so as to mimic the patient LBC situation(Figure 4A), we found that modestly reduced CHD1Lwas indeed associated with impaired DCC activation fol-lowing Topo II inhibition and resulted in increase innumber of pseudomitoses (Figure 4B). These datadescribe a novel consequence of limiting CHD1L levels.Failure of the DCC can also ultimately result in chro-mosome breakage and elevated levels of genomicinstability as evidenced by increase in micronuclei[30,31]. Consistent with DDC failure observed in 1q21.1Del and Dup containing LBCs, we found elevated levelsof micronuclei in both LBCs following prolonged treat-ment (16 hrs) with ICRF193, although to a greaterextent in the 1q21.1 Del containing LBCs compared tothe 1q21.1 Dup containing LBCs (Figure 4C). Neverthe-less, these data are consistent with a failure to efficientlyactivate the DCC and with elevated levels of DSBswhich manifest as micronuclei in these cultures (Figure4C). There was no evidence of spontaneous chromo-some instability or increased micronuclei formation inthe 1q21.1 Del and 1q21.1 Dup containing cell linesbased on analysis of solid stained and G banded patientchromosomes and nuclei after short term culture.Functional assays for PRKAB2AMP-activated protein kinase (AMPK) senses and regu-lates systemic and cellular energy balance by regulatingHarvard et al. Orphanet Journal of Rare Diseases 2011, 6:54http://www.ojrd.com/content/6/1/54Page 6 of 12food intake, body weight, and glucose and lipid homeos-tasis [32]. It also plays an important role in negativelyregulating the mTOR pathway that functions to controlribosome and protein biosynthesis [33]. AMPK is a het-erotrimeric complex composed of a catalytic a-subunit,a regulatory b-subunit and an ADP/ATP-binding g-sub-unit [34]. Furthermore, several isoforms of each subunitexist (a1, a2, b1, b2, g1, g2, g3) thereby enabling thegeneration of multiple distinct heterotrimeric complexes.PRKAB2 encodes the b2-isoform of AMPK.Expression of PRKAB2 protein product, AMPKb2, inpatient cells was decreased in the cell line with 1q21.1Del and increased in the cell line with 1q21.1 Dup com-pared to a wild-type (WT) control, whilst that of the b1subunit was unaffected (Figure 5A and 5B). The geneencoding AMPK-b1 subunit (PRKAB1) is located onchromosome 12q24.1 and so is not within the 1q21.1CNV. To investigate the impact of increased anddecreased AMPK-b2 expression on AMPK activity wetreated patient-derived LBCs with AICAR (N1-(b-D-Ribofuranosyl)-5-aminoimidazole-4-carboxamide), a cellpermeable nucleoside analogue that mimics the effectsof AMP on the allosteric activation of AMPK, and mon-itored phosphorylation of AMPK on threonine-172 (p-Figure 3 Functional assays for CHD1L in patient cells. (A) Left panels: Western analysis of CHD1L expression from wild-type (WT), 1q21.1deletion (Del) and duplication (Dup) LBCs following titration of whole cell extracts. Right Panels: b-tubulin re-probe to confirm equal loading.(B). Densiometric quantification of CHD1L expression from Western analysis from low (black bar), intermediate (white) to higher (grey) amountsof protein, from each line, using three separate determinations, normalized to b-tubulin loading (a.u. arbitrary units). p = 0.009 for Del and p <0.005 for Dup compared to WT. (C). The Decatenation Checkpoint (DCC). Unreplicated DNA sequences between converging replication forksundergo catenation and torsional tension which is normally relieved by Topoisomerase IIa (Topo IIa) which induces a transient DSB enablingdecatenation (untangling). DCC activation in G2 prevents entry into mitosis until sister chromatids are fully separated. DCC can be activated byTopo II inhibitors arresting the cycle in G2 (indicated in red). DCC failure is monitored by the enumeration of ‘pseudomitosis’ containing highlycatenated (entangled) chromatids. Inset image shows typical pseudomitotic cells following treatment of the Del LBCs with the Topo II inhibitor,ICRF-193. (D). Mitotic index (Mitosis %) and Pseudomitotic index (Pseudomitosis %) for untreated (Unt) LBCs or ICRF-193 treated, for wild-type(WT), Werner’s syndrome (WRN), Dup and Del containing LBCs. WRN LBCs are known to be defective in DCC activation. Data presented indicatesthe mean ± s.d of three separate determinations. p < 0.005 for reduction in Mitosis (%) Unt compared to ICRF-193 and p < 0.005 for increase inPseudomitosis (%) Unt compared to ICRF-193.Harvard et al. Orphanet Journal of Rare Diseases 2011, 6:54http://www.ojrd.com/content/6/1/54Page 7 of 12T172-AMPKa). This is an essential modification,required for and diagnostic of AMPK activity (Figure5C). Interestingly, both the 1q21.1 Dup and 1q21.1 Delcontaining LBCs exhibited slightly elevated basal levelsof p-T172-AMPKa in the absence of AICAR (0 time),compared to wild-type (WT). Elevated AICAR-inducedp-T172-AMPKa was detectable in wild-type LBCs (WT)within 5 minutes, and to a similar extent 1q21.1 Dupcontaining cells (Figure 5C). In comparison, the changein the AICAR-induced p-T172-AMPKa activity at 5minutes was less apparent in the 1q21.1 Del containingcell line, and the activity remained constant at 15 min-utes. In contrast, the AICAR-induced p-T172-AMPKaactivity of the WT and 1q21.1 Dup containing cell linewas reduced after 15 minutes. This suggests thatdecreased AMPK-b2 level is associated with somewhatunresponsive AMPK activation, while the 1q21.1 dupli-cation-containing LBC (Dup) showed similar pattern ofresponsiveness to WT cells under these conditions (Fig-ure 5C).To further substantiate these findings we exploredAMPK-mediated phosphorylation of two of its wellknown substrates, Acetyl-CoA Carboxylase (ACC)and RAPTOR. ACC is a key mediator of fatty acid(FA) synthesis. AMPK-induced phosphorylation ofACC on serine-79 (p-S79-ACC) inhibits ACC enzy-matic activity thereby limiting FA synthesis underenergy limiting conditions (i.e. high [AMP] and low[ATP]) [32]. Consistent with our findings with p-T172-AMPKa, we found efficient induction of p-S79-ACC in WT and LBCs with 1q21.1 Dup within 5minutes of AICAR treatment, whilst the LBCs with1q21.1 Del failed to exhibit significant levels of p-S79-ACC under these conditions. This data supportsthe observation of sub-optimal AMPK activity in thisline (Figure 5D).Figure 4 Consequences of limiting CHD1L levels by siRNA. (A). Careful titration of CHD1L siRNA conditions were undertaken in A549 so asto mimic haploinsufficient expression of CHD1L. Left panels:These show the Western analysis of whole cell extracts from Untreated (Unt; mock-treated) control whereas siRNA indicates cells treated with CHD1L siRNA. b-tubulin expression was monitored to confirm equal loading. Rightgraph: Densiometric quantification of CHD1L expression, normalized to b-tubulin loading from three separate siRNA experiments. The degree ofCHD1L reduction is very similar to that observed from the 1q21.1 deletion (Del) containing LBC (Fig 3A and B). Data represents the mean ± s.d.of three separate experiments (a.u. arbitrary units). (B). Inset image shows a typical catenated pseudomitotic cell following CHD1L siRNA-mediated knockdown in A549 treated with Topo II inhibitor (ICRF-193). The % pseudomitosis were enumerated under various conditions in A549following CHD1L siRNA-mediated knockdown to mimic haploinsufficiency. Unt (untreated; not treated with ICRF-193), ICRF-193 (treated withICRF-193), Con (control siRNA scrambled oligo), CHD1L siRNA (treated with siRNA to mimic CHD1L haploinsufficiency). Data represents the mean± s.d. of three separate experiments and p < 0.005 for increase in Pseudomitosis (%) following CHD1L siRNA. (C). Inset image shows micronuclei(MN). The % of ICRF-193-induced MN in binucleate cells were determined in wild type (WT), Dup and Del containing LBLs following a 24 hrrecovery from an overnight treatment with ICRF-193. Data represents the mean ± s.d. of three separate experiments and p = 0.02 for increase %MN in binucleates for Dup and p < 0.005 for Del containing LBCs.Harvard et al. Orphanet Journal of Rare Diseases 2011, 6:54http://www.ojrd.com/content/6/1/54Page 8 of 12RAPTOR is an important regulatory component of themTOR containing complex 1 (mTORC1) and isrequired for optimal mTOR kinase activity [35]. AMPK-mediated phosphorylation of RAPTOR on serine-792(p-S792-RAP) inhibits mTORC1 thereby limiting proteinsynthesis and inducing cell cycle arrest when cellularenergy is limiting. Again, consistent with sub-optimalAMPK activity in the 1q21.1 Del containing LBCs, wefound reduced AICAR-induced p-S792-RAPTOR inthese cells in contrast to the 1q21.1 Dup containing lineand the WT control (Figure 5D). Together, these resultssuggest that haploinsufficiency of PRKAB2 results inreduced expression of AMPK-b2 which is associatedwith impaired AICAR-induced AMPK activation. Incontrast, duplication of PRKAB2 did not negativelyimpact on AMPK activity under the conditions exam-ined here.DiscussionWe have performed whole genome expression and cellfunction studies in carriers of 1q21.1 deletion and1q21.1 duplication. Our data show that the top genesranked based on correlation of expression and 1q21.1copy number change are significantly enriched for1q21.1 genes, indicating association of expression andcopy number for ~50% of 1q21.1 CNV genes. Further-more, we show that the function of proteins coded bytwo of the genes from the 1q21.1 CNV, which rankedFigure 5 Functional assays for PRKAB2 in patient cells. (A). Left panels:Titrated whole cell extracts wereblotted for AMPKb2 (encoded byPRKAB2) in wild-type (WT),Del and Dup containing LBCs. Right panels:Blots were re-probed with anti-b-tubulin. Graph: Densiometricquantification of AMPK-b2 expression from titrated extracts, going from low (black bar), intermediate (white) to higher (grey) amounts of protein,normalized to b-tubulin loading, from three separate determinations (a.u. arbitrary units). p = 0.01 for Del and p < 0.005 for Dup LBCs comparedto WT. (B). AMPK subunit AMPK-b1, encoded by the PRKAB1 gene on chromosome 12q24.1, shows equivalent expression in the wild-type (WT),Del and Dup containing LBCs. b-tubulin was used to confirm equal loading. (C). AICAR-induced (2 mM) activation of the AMPK kinase wasmonitored using phosphorylation of the AMPKa subunit on threonine 172 (p-T172-AMPKa). Dup and Del containing LBCs exhibited higherlevels of p-T172-AMPKa at the 0 time (untreated), relative to wild-type (WT). Only the 1q21.1 Del containing LBCs appeared to be unresponsiveto AICAR-treatment here. Blots were re-probed with for native AMPKa to confirm loading. (D). AICAR-induced (2 mM) activation of AMPK wasevaluated by monitoring phosphorylation of the AMPK substrate Acetyl-CoA Carboxylase on serine 79 (p-S79-ACC). Similar to p-T172-AMPKa, theDel containing LBCs appear unresponsive to the AICAR treatment. Blots re-probed for native ACC to confirm loading. (E). AICAR-inducedactivation of AMPK was also evaluated by phosphorylation of the AMPK substrate RAPTOR on serine 792 (p-S792-RAP) under identical conditionsas in (B) and (C). Again, Del containing LBCs appeared somewhat unresponsive to AICAR. Blots re-probed for MCM2 to confirm loading.Harvard et al. Orphanet Journal of Rare Diseases 2011, 6:54http://www.ojrd.com/content/6/1/54Page 9 of 12highest in 1q21.1 copy number expression correlation, isaltered in both the deletion and duplication patient celllines.CHD1L, the gene which ranked first in the expression/1q21.1 copy number correlation, has been implicated inchromatin remodeling and relaxation as well as DNAdamage response [25,26]. Our studies identified a novelrole for CHD1L in decatenation, which was suspectedbased on its known chromatin remodeling function, andthe defective Topo II decatenation checkpoint demon-strated here in both the 1q21.1 Del and Dup containingpatient cell lines.It is of interest that the DCC defect detected in the1q21.1 Del and Dup containing cell lines is comparableto that seen in cells from Werner syndrome (OMIM277700), an autosomal recessive disorder, associatedwith predisposition to cancer and premature aging,neither of which were noted in our patients. The onlyoverlapping feature, short stature, was noted in 5/6 sub-jects with the 1q21.1 deletion and also reported in sub-jects from other cohorts [2]. Previous DCC studies ofWerner syndrome and control cells suggested that DCCdefect per se is not sufficient to cause significant geno-mic instability, but requires absence or dysfunction of“caretaker” genes such as ATR, BRCA1 or WRN [29]. Itis possible that in cell lines with 1q21.1 Del and Dupthe DCC defect is not accompanied with other deleter-ious events and thus the threshold for significant spon-taneous genomic instability leading to premature cellsenescence/cancer predisposition is not met. We havenot found evidence of spontaneous chromosomeinstability in the short term chromosome cultures ofour patients nor has this been previously reported forany of the1q21.1 CNV subjects who had routine chro-mosome analysis. Future studies of the association ofCHD1L with other genes in decatenation checkpointmechanism may shed more light on the precise role ofCHD1L in DCC. So, whilst the phenotypic consequencesof defective DCC activation in subjects with a 1q21CNV are unclear, their cellular phenotype does appearto be consistent with CHD1L dysfunction.Our findings that the same cellular phenotype is pre-sent in both the 1q2.1 Del and Dup containing celllines, is in keeping with reports [26] that both dosageimbalances of CHD1L result in identical cellular effects.Haploinsufficiency and duplication sensitivity is thoughtto affect genes regulating balanced expression of othergenes ("master genes” [36]), which is in keeping withCHD1L’s role as a chromatin remodeler and indirectregulator of many key biological processes such as repli-cation, transcription and translation [37]. In that respect,it is interesting to note that > 18 genes with a role inchromatin remodeling have been implicated in intellec-tual disability [38].PRKAB2, which ranked 10th in the expression/1q21.1copy number correlation, encodes the b2-subunit ofAMPK, a key regulator of cellular response to a largenumber of external stimuli which modulates energylevels at the cellular and organism level [39]. The dereg-ulation of AMPKb2 function in 1q21.1 deletion andduplication carriers was suspected based on a) changesin levels of AMPKb2 protein (in keeping with the1q21.1 copy number state and expression level of thePRKAB2 gene), b) different basal levels of p-T172-AMPKa in both 1q21.1 Del and Dup containing lines incomparison to WT, and c) sub-optimal AICAR-inducedphosphorylation of the AMPK substrates ACC andRAPTOR, which was more obvious in the 1q21.1 Delcontaining line. The last observation could be explainedby the fact that AMPK, as a multi protein complex, maybe sensitive to imbalances of its components [36], andthat reduced availability of a regulatory b-isoform, asoccurs here, could impact on AMPK activity more thanover-abundance.The multifaceted nature of AMPK role in brain func-tion is of particular interest to the 1q21.1 phenotypewhich most consistently includes some form of learningdifficulty. Previous studies showed that alternations ofAMPK activity resulted in profound abnormalities of thecentral nervous system in AMPK-b1-/- knockout micewhich had reduction of AMPK activity [34], whereas theconsequences of AMPK activation remain controversialas some groups have shown that AMPK activation isneuroprotective while others show that AMPK overacti-vation is detrimental [39]. The essential role of AMPKin brain function is further supported by its inhibitionof the mTOR pathway [32] which is required for learn-ing and memory [40].Our studies are the first to report that the function oftwo genes integral to 1q21.1 CNV is changed in patientlymphoblasts and that both genes are likely to be dosagesensitive. Both genes are expressed in multiple tissues,including brain [34,41] which may explain the multi-sys-temic nature of the physical abnormalities, and the fre-quent involvement of learning difficulty albeit at a veryvariable levels. It remains uncertain as to the tissue-spe-cific consequences of gene function changes in indivi-duals with 1q21.1 CNV although AMPK is clearlyinvolved in brain development and homeostasis. Webelieve that our investigations are unique as theypointed to genes for which further functional investiga-tion in additional carriers and cell lines may prove to beworthwhile.The phenotypic variability for some CNVs has beentraditionally explained by genetic and environmentalfactors [42]. In that respect it is of interest to note thatCHD1L and PRKAB2 have a role in sensing andresponding to genomic (chromosomal structure) andHarvard et al. Orphanet Journal of Rare Diseases 2011, 6:54http://www.ojrd.com/content/6/1/54Page 10 of 12metabolic (energy level) stress and therefore their dys-function may result in a more severe phenotype in indi-viduals which experienced more adverse environmentalconditions during development. Sequence changes ofother genes from the 1q21.1 region as well as othergenes across the genome that impair their function can-not be ruled out as a source of variability at this timeand the new whole genome sequencing technologies willno doubt become useful in future investigations of theircontribution to the development of an abnormalphenotype.ConclusionOur studies are unique as they provide evidence ofchanges in the function of genes from 1q21.1 CNV inlymphoblasts from both deletion and duplication car-riers. Furthermore, they also provide evidence that dele-tions and duplications can have similar (e.g. DCCdeficiency in 1q21.1 Del and Dup containing LBCs) aswell as differing functional consequences (e.g. lessresponsive AICAR-induced AMPK activity in 1q21.1 Delcontaining LBCs) depending on the genes and pathwaysinvolved. Our results support the use of patient lympho-blasts for dissecting the functional sequelae of genesintegral to CNVs in carrier cell lines, ultimately enhan-cing understanding of biological processes which maycontribute to the clinical phenotype.Additional materialAdditional file 1: Table S1: Clinical and genomic information onsubjects included in the study. Table S2: Top 100 Genes fromExpression/1q21.1 Copy Number correlation analysis.Additional file 2: Figure S1: Correlation of expression and 1q21.1copy number for probes across the genome expressed as log10 ofp values.AcknowledgementsWe are grateful to the subjects’ and their extended family members’enthusiastic support of this study. This work was supported by a CIHRoperating grant (MOP 74502; PI: ERS) and Career Investigator Award fromthe Michael Smith Foundation for Health Research (ERS, PP and MESL). TheM.O’D lab is funded by Cancer Research UK and the UK Medical ResearchCouncil. M.O’D is a CRUK Senior Cancer Research Fellow.Author details1Child and Family Research Institute, Molecular Cytogenetics and ArrayLaboratory, 950 West 28th Avenue, Vancouver, BC, Canada. 2Department ofPathology, University of British Columbia, Vancouver, BC, Canada.3Department of Psychiatry, University of British Columbia, Vancouver, BC,Canada. 4Human DNA Damage Response Disorders Group, Genome Damage& Stability Centre, University of Sussex, Brighton, UK. 5Clinical GeneticsService, Centre for Addiction and Mental Health, Toronto, and Departmentof Psychiatry, University of Toronto, Canada. 6Cytogenetics Laboratory, RoyalColumbian Hospital, New Westminster, BC, Canada. 7Department of MedicalGenetics, University of British Columbia, Vancouver, BC, Canada.8Departments of Pediatrics and Child Health and Biochemistry and MedicalGenetics, University of Manitoba, Winnipeg, MB, Canada. 9Department ofPhysiology, Queen’s University, Kingston, Ontario, Canada. 10Department ofPsychiatry, Queen’s University, Kingston, Ontario, Canada. 11Genetics andGenomics Research Laboratory, Ongwanada, Kingston, Ontario, Canada.Authors’ contributionsERS designed the study, initiated the collaborative project, monitored datacollection for the whole study, and revised the paper. M.O’D designed thefunctional analysis for the two genes, and wrote the functional aspect of thepaper. ES and CH performed the genomic and expression relatedexperiments and drafted the manuscript. SMartell and YQ contributed togenomic array data analysis. RC and DA performed the functional studies.ME and PP performed the statistical analysis of the expression data. EC, SH,BM, SMarles, AM, AD, SL contributed with clinical information on thesubjects. JJA reviewed the manuscript. MH and CT supervised the 2.7Affymetix genomic analysis. All authors approved and read the finalmanuscript.Competing interestsThe authors declare that they have no competing interests.Received: 10 April 2011 Accepted: 8 August 2011Published: 8 August 2011References1. 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