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Detection of pathogenic copy number variants in children with idiopathic intellectual disability using… Friedman, JM; Adam, Shelin; Arbour, Laura; Armstrong, Linlea; Baross, Agnes; Birch, Patricia; Boerkoel, Cornelius; Chan, Susanna; Chai, David; Delaney, Allen D; Flibotte, Stephane; Gibson, William T; Langlois, Sylvie; Lemyre, Emmanuelle; Li, H I; MacLeod, Patrick; Mathers, Joan; Michaud, Jacques L; McGillivray, Barbara C; Patel, Millan S; Qian, Hong; Rouleau, Guy A; Van Allen, Margot I; Yong, Siu-Li; Zahir, Farah R; Eydoux, Patrice; Marra, Marco A Nov 16, 2009

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ralssBioMed CentBMC GenomicsOpen AcceResearch articleDetection of pathogenic copy number variants in children with idiopathic intellectual disability using 500 K SNP array genomic hybridizationJM Friedman*1,2, Shelin Adam1, Laura Arbour1,3, Linlea Armstrong1,4, Agnes Baross5, Patricia Birch1, Cornelius Boerkoel1,2,4, Susanna Chan5, David Chai1,4, Allen D Delaney5, Stephane Flibotte5, William T Gibson1,2,4, Sylvie Langlois1,4, Emmanuelle Lemyre6, H Irene Li5, Patrick MacLeod3, Joan Mathers4, Jacques L Michaud7, Barbara C McGillivray1,4, Millan S Patel1,4, Hong Qian5, Guy A Rouleau7, Margot I Van Allen1,4, Siu-Li Yong1,4, Farah R Zahir1, Patrice Eydoux4 and Marco A Marra1,5Address: 1Department of Medical Genetics, University of British Columbia, Vancouver, Canada, 2Child & Family Research Institute, Vancouver, British Columbia, Canada, 3Victoria General Hospital, Victoria, British Columbia, Canada, 4Children's & Women's Health Centre, Vancouver, British Columbia, Canada, 5Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, Canada, 6CHU Sainte-Justine Research Center, Montréal, Québec, Canada and 7Center for Excellence in Neuromics of Université de Montréal, CHU Sainte-Justine Research Center, Montréal, Québec, CanadaEmail: JM Friedman* - frid@interchange.ubc.ca; Shelin Adam - shelinadam@gmail.com; Laura Arbour - Laura.Arbour@viha.ca; Linlea Armstrong - llarmstrong@cw.bc.ca; Agnes Baross - abaross@genomebc.ca; Patricia Birch - birch@interchange.ubc.ca; Cornelius Boerkoel - boerkoel@interchange.ubc.ca; Susanna Chan - schan@bcgsc.ca; David Chai - dchai33@hotmail.com; Allen D Delaney - adelaney@bcgsc.ca; Stephane Flibotte - sflibotte@bcgsc.ca; William T Gibson - wtgibson@cmmt.ubc.ca; Sylvie Langlois - SLANGLOIS@cw.bc.ca; Emmanuelle Lemyre - emmanuelle.lemyre@recherche-ste-justine.qc.ca; H Irene Li - ili@bcgsc.ca; Patrick MacLeod - patrick.macleod@viha.ca; Joan Mathers - JMathers@cw.bc.ca; Jacques L Michaud - jacques.michaud@recherche-ste-justine.qc.ca; Barbara C McGillivray - bmcgillivray@cw.bc.ca; Millan S Patel - mPatel@cw.bc.ca; Hong Qian - hqian@bcgsc.ca; Guy A Rouleau - guy.rouleau@umontreal.ca; Margot I Van Allen - mvallen@cw.bc.ca; Siu-Li Yong - slyong@cw.bc.ca; Farah R Zahir - farahz@interchange.ubc.ca; Patrice Eydoux - PEydoux@cw.bc.ca; Marco A Marra - mmarra@bcgsc.bc.ca* Corresponding author    AbstractBackground: Array genomic hybridization is being used clinically to detect pathogenic copynumber variants in children with intellectual disability and other birth defects. However, there isno agreement regarding the kind of array, the distribution of probes across the genome, or theresolution that is most appropriate for clinical use.Results: We performed 500 K Affymetrix GeneChip® array genomic hybridization in 100idiopathic intellectual disability trios, each comprised of a child with intellectual disability ofunknown cause and both unaffected parents. We found pathogenic genomic imbalance in 16 ofthese 100 individuals with idiopathic intellectual disability. In comparison, we had found pathogenicgenomic imbalance in 11 of 100 children with idiopathic intellectual disability in a previous cohortPublished: 16 November 2009BMC Genomics 2009, 10:526 doi:10.1186/1471-2164-10-526Received: 15 April 2009Accepted: 16 November 2009This article is available from: http://www.biomedcentral.com/1471-2164/10/526© 2009 Friedman et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 20(page number not for citation purposes)who had been studied by 100 K GeneChip® array genomic hybridization. Among 54 intellectualdisability trios selected from the previous cohort who were re-tested with 500 K GeneChip® arrayBMC Genomics 2009, 10:526 http://www.biomedcentral.com/1471-2164/10/526genomic hybridization, we identified all 10 previously-detected pathogenic genomic alterations andat least one additional pathogenic copy number variant that had not been detected with 100 KGeneChip® array genomic hybridization. Many benign copy number variants, including one that wasde novo, were also detected with 500 K array genomic hybridization, but it was possible todistinguish the benign and pathogenic copy number variants with confidence in all but 3 (1.9%) ofthe 154 intellectual disability trios studied.Conclusion: Affymetrix GeneChip® 500 K array genomic hybridization detected pathogenicgenomic imbalance in 10 of 10 patients with idiopathic developmental disability in whom 100 KGeneChip® array genomic hybridization had found genomic imbalance, 1 of 44 patients in whom100 K GeneChip® array genomic hybridization had found no abnormality, and 16 of 100 patientswho had not previously been tested. Effective clinical interpretation of these studies requiresconsiderable skill and experience.BackgroundChromosomal imbalance has been recognized as themost frequent cause of intellectual disability (ID) for 50years [1-3]. Until recently, most of this genomic imbal-ance was diagnosed by cytogenetic analysis, but studiesover the past few years have found that ID is caused byconstitutional gains or losses of submicroscopic genomicsegments even more often than by microscopically-appar-ent chromosomal aberrations [4-6]. Our ability to recog-nize these submicroscopic genomic changes, which areusually called copy number variants (CNVs), as the mostfrequent cause of ID depends on the use of Array GenomicHybridization (AGH) (also known as array-comparativegenomic hybridization, chromosomal microarray analy-sis, or copy number analysis). AGH can survey the entiregenome for imbalance that is 1/100th the size of thatdetectable by conventional cytogenetic analysis.Although AGH is now being used routinely as a clinicaltest for the identification of chromosomal imbalance inpeople with ID and other birth defects, controversy stillexists regarding the most appropriate platform to use forthis purpose. Initial clinical studies were done with arrayshaving a few thousand BACs distributed at 1-3 Mb inter-vals across the genome or with BACs targeted to regionswhere pathogenic submicroscopic deletions or duplica-tions were known to occur. More recent studies haveshown that arrays with higher resolution and genome-wide coverage provide better detection rates for patho-genic CNVs in children with ID and normal cytogeneticanalysis [7-12]. Other methods have identified patho-genic CNVs that are too small to have been detected by thearray platforms used in most AGH studies [13-15], soanalysis at even higher resolution may be necessary todetect all pathogenic CNVs in children with ID.Unfortunately, use of higher resolution AGH for detectionof genomic imbalance is confounded because most CNVsvarious studies, depending on the technology used toidentify the variants and the size range used to define aCNV [16-25]. Sequencing of the complete diploidgenomes of normal individuals has shown that thenumber of CNVs per person is actually even greater [26-29]. Distinguishing these benign CNVs from those thatcause ID and other birth defects is the most serious chal-lenge to routine clinical use of AGH.We previously reported our findings using 100 K Affyme-trix GeneChip® AGH [10,16] to perform a genome-widesurvey of benign and pathogenic CNVs in 100 idiopathicID trios, each comprised of an affected child and bothunaffected parents. Here we describe the results of a studyof 100 new idiopathic ID trios, as well as 54 of the triostested previously, using 500 K Affymetrix GeneChip®AGH. We found that higher resolution AGH detected alarger number of apparently pathogenic CNVs in bothgroups. Many benign CNVs, including at least one thatwas de novo, were also detected with the 500 K AGH, butit was possible to distinguish benign and pathogenicCNVs with confidence in almost all cases.ResultsWe performed AGH with 500 K Affymetrix GeneChip®arrays on 154 children with idiopathic ID and on bothparents of each affected child. Fifty-four of these trios(called "the 100 K cohort") had previously been studiedwith lower-resolution 100 K GeneChip® AGH [10,16]; theother 100 trios (called "the new cohort") were studied byAGH for the first time with the 500 K GeneChip® arrays.Data were analyzed to determine copy number along thelength of all chromosomes except the Y (for which thereare no probes on either the 100 K or 500 K GeneChip®arrays), and the findings for each child were comparedwith those for his or her parents. Autosomal CNVs seen inthe child and in at least one parent were considered likelyto be benign polymorphisms. Autosomal CNVs found inPage 2 of 20(page number not for citation purposes)are not pathogenic. Estimates of the mean number ofapparently benign CNVs per person range from 6-824 inthe child but not in either parent were evaluated by anindependent method to confirm the presence of the CNVBMC Genomics 2009, 10:526 http://www.biomedcentral.com/1471-2164/10/526and its de novo occurrence. CNV calls on the X-chromo-some in a female child were validated by an independentmethod if they appeared to have occurred de novo; CNVcalls on the X-chromosome in a male child were validatedby an independent method whether they appeared to bede novo or to have been inherited from the mother.We found a total of 4577 hits (putative CNVs including atleast 10 contiguous SNPs called by the SMD software witha p-value below 1 × 10-8) in the 462 samples (154 trios)analyzed by 500 K GeneChip® AGH. This is an average ofabout 10 hits per sample, which is an underestimate of thetotal number of benign CNVs present because of the strin-gent cutoff used to obtain a false discovery rate of less than5% (see Methods).Within the 54 trios who were studied with both platforms,we found four times as many hits with 500 K AGH as wedid with 100 K AGH (Table 1). The ratio of the number ofputative CNVs called with the 500 K platform to thenumber called with the 100 K platform was 11.0 for CNVsbetween 100 kb and 200 kb in size but was lower for bothsmaller and larger CNVs (4.1 for those < 100 kb and 3.4for those between 200 kb and 500 kb).The 4577 putative CNVs called in all 154 trios were sub-jected to further bioinformatic analysis (see Methods) toproduce a final annotated list of 58 apparently de novoCNVs and two cases of mosaic trisomy that were called in50 patients. These apparent genomic imbalances weresubjected to validation by independent methods. Thus, anapparent de novo CNV call that required independent val-idation was made in about 1 child in 3. Thirty-three ofthese CNVs in 30 patients and both cases of mosaic tri-somy were confirmed to be de novo by an independentmethod and are described in detail below. The other puta-tive CNVs were found to be present in both of the parentsas well as in the child (false negative AGH calls in the par-ents) in two instances or could not be confirmed to bepresent in the child (false positive AGH calls) in 22instances. Altogether, false positive CNV calls were madein 21 (13.6%) of the 154 trios studied and false negativeCNV calls were made in 2 (1.3%) of the 154 trios studied.In one other trio (Family 5202), an apparent de novo dele-tion of chromosome 14 called on AGH in the child wasfound by FISH to be a duplication of the region in bothparents instead.Nineteen of 100 children with ID in the new cohort werefound by Affymetrix 500 K GeneChip® AGH to have deTable 1: Putative CNVs called on 100 K and 500 K AGH in 54 trios tested with both technologies.Comparison Child to Father Child to MotherArray 100 K 500 K 100 K 500 KAll CNV Calls Number 125 497 110 501Number per Child 2.31 9.20 2.04 9.28Statistical Significance t = 7.18, p < 0.0001 t = 6.67, p < 0.0001CNV Calls < 100 kb Number 59 237 53 221Number per Child 1.06 4.39 0.98 4.09Statistical Significance t = 7.82, p < 0.0001 t = 5.19, p < 0.0001CNV Calls ≥ 100 kb and < 200 kb Number 13 130 12 146Number per Child 0.24 2.41 0.22 2.70Statistical Significance t = 5.39, p < 0.0001 t = 6.27, p < 0.0001CNV Calls ≥ 200 kb and < 500 kb Number 33 101 25 98Number per Child 0.61 1.87 0.46 1.81Statistical Significance t = 4.66, p < 0.0001 t = 5.25, p < 0.0001Page 3 of 20(page number not for citation purposes)Two-tailed statistical significance was calculated with Student's t-test.BMC Genomics 2009, 10:526 http://www.biomedcentral.com/1471-2164/10/526novo genomic imbalance that was confirmed by FISH,MLPA, AGH on an Agilent® 244 K platform or cytogeneticre-analysis (Table 2). One of these children (Patient 8056)had mosaic trisomy 9, and two were found to have de novounbalanced reciprocal translocations - a der(10)t(2;10)(q37;q26.13) in Patient 873 and a der(4)t(4;8)(p16.1;p23.1) in Patient 5814 - each producing botha terminal duplication and a terminal deletion identifiedby AGH. We found de novo submicroscopic deletions in13 other patients and de novo submicroscopic duplica-tions in three other patients. The deletions ranged in sizefrom 89 kb to 11.0 Mb; six were less than 1 Mb. The dupli-cations ranged in size from 362 kb to 11.1 Mb; one wasless than 1 Mb.Using 500 K Affymetrix GeneChip® AGH, we also con-firmed the genomic imbalance that had previously beenidentified in 10 of the 54 ID trios from the 100 K cohort(Table 3 and Additional File 1: Supplemental Table S1). Inaddition, we identified and confirmed by FISH two denovo CNVs that were not called on the 100 K assay - a 1.6Mb duplication of 8q23.2-23.3 in Patient 3890 and a 1.5Mb deletion of 4p16.3 in Patient 4840 (Table 3 and Figure1).We found two instances of uniparental disomy (UPD),diagnosed by the occurrence of mendelian inconsistencyin a region with a normal copy number of 2 [30], amongthe 100 ID trios in the new cohort studied by 500 KAffymetrix GeneChip® AGH (Table 4 and Figures 2A and2B). Patient 6904 has mosaic paternal isodisomy of mostof the short arm of chromosome 11. Patient 1658 hasmaternal UPD 16, being heterodisomic for the centralportion of the chromosome and isodisomic for both ends.Both cases were confirmed to be disomic with microsatel-lite markers (Figures 2C and 2D).We judged the mosaic trisomy 9, both unbalanced recip-rocal translocations, 11 of the other de novo deletions, andtwo of the other de novo duplications found in the newcohort to be pathogenic (Table 2). A mosaic 107 kb denovo deletion of chromosome 14 q11.2 (Patient 818) anda homozygous 89 kb deletion of the HLA-G region thatresulted from transmission by parents who both carriedheterozygous deletions of the same region (Patient 216)were judged to be benign variants. A 186 kb deletion ofchromosome 21q22.11 (Patient 8619), a 362 kb duplica-tion of chromosome 22 q11.21 (Patient 9979) and bothcases of UPD (Patients 1658 and 6904) were of uncertainclinical significance.Of the two de novo CNVs identified by 500 K AffymetrixGeneChip® AGH in the 44 children with ID whose 100 K® of uncertain clinical significance. A 1.5 Mb deletion of4p16.3 in Patient 4840 is pathogenic because of its size,the inclusion of two genes that have been implicated inthe Wolf-Hirschhorn syndrome (WHSC1 and WHSC2),overlap with known pathogenic CNVs, and a compatibleclinical phenotype. The 1.6 Mb duplication of 8q23.2-23.3 in Patient 3890 is of unknown clinical significance.It includes no RefSeq genes but involves a large (1.6 Mb)genomic region, most of which has never been reported tobe polymorphic in normal individuals.DiscussionBecause its detection rate for pathogenic genomic imbal-ance is much higher than that of conventional cytogeneticanalysis, a consensus has developed that AGH should beused clinically for the evaluation of patients with ID andother birth defects [31-38]. It is clear that AGH using "tar-geted" arrays that only include probes for genomic regionsknown to be involved in microdeletion or microduplica-tion syndromes has substantially lower detection rates forCNVs that cause ID than AGH using arrays that providegenome-wide coverage [37,39-41]. Beyond this, however,there is no agreement regarding the kind of array, the dis-tribution of probes across the genome, or the resolutionthat is most appropriate for clinical use. Although BACarrays were initially used, most clinical laboratories nowprefer oligonucleotide arrays because high-quality plat-forms that produce consistent results are reliably availablefrom commercial sources. In addition, the use of largernumbers of smaller probes on oligonucleotide arrays per-mits more precise delineation of the breakpoints of CNVsthat are detected, which facilitates genotype-phenotypecorrelation and clinical interpretation. AGH with a SNParray provides the additional advantage of generating gen-otypes that can be used to verify family relationships andfind uniparental disomy as well as a second method (inaddition to hybridization intensity) for identifyinggenomic imbalance [10,42-44].We previously reported that 100 K Affymetrix GeneChip®AGH is a robust platform for the detection of pathogenicCNVs in patients with ID [10]. Here we show that thedetection rate of CNVs among such patients is higher with500 K GeneChip® AGH than with 100 K GeneChip® AGH.We made about four times as many CNV calls overall withthe 500 K platform as with the 100 K platform when usingthe same method of bioinformatic analysis in 54 triosstudied with both technologies (Table 1). We also found18 instances of pathogenic genomic imbalance in 16 of100 children with ID and normal cytogenetic analysisstudied by 500 K GeneChip® AGH (Table 2), compared to11 instances of pathogenic genomic imbalance in 11 of100 similarly-ascertained children tested by 100 K Gene-® Page 4 of 20(page number not for citation purposes)GeneChip AGH studies had been interpreted as normal(Table 3), one is likely to be pathogenic and the other isChip AGH in our previous study. Although the higherdetection rate we observed with the 500 K platform mayBMC Genomics 2009, 10:526http://www.biomedcentral.com/1471-2164/10/526Page 5 of 20(page number not for citation purposes)RefSeq Genes Involved*Comments Interpretation~70 genes including AGRN, GNB1, PEX10, PRKCZ, SKI, and TP73This CNV is included in the 1p36 deletion syndrome critical region, and the patient's clinical features are compatible with that syndrome [73].Pathogenic~100 genes including AGXT, ATG16L1, CAPN10, CHRND, CHRNG, COL6A3, D2HGDH, GBX2, DAC4, MLPH, PDCD1, ER2, SAG and UGT1A1Pathogenic~75 genes including ADAM12, CTBP2, OCK1, DPYSL4, FGFR2, OAT, and UROSTable 2: Genomic imbalance and uniparental disomy detected by 500K GeneChip® AGH in the new cohort.Patient Change Location SNP CountStart→EndSize (bp) Validation Phenotype9133 Deletion 1p36.32-p36.33198 769,185→ 3,581,3082,812,123 FISH, MLPA 9 year-old female with obesity, moderate cognitive impairment, myoclonus, polyphagia, hypotonia, narrow frontal area, deep-set eyes, prominent orbital rims, short nose with low nasal bridge and upturned nasal tip, midface retrusion, short philtrum, tented upper lip, thoracic kyphosis, small distal phalanges of the toes, strabismus, and 11 ribs873 Duplication†2q37 1801 231,577,285→ 242,663,30311,086,018 FISH, MLPA 15 year-old male with severe cognitive impairment, birth weight < 1st centile, birth length < 1st centile, head circumference at birth < < 10th centile, hypotonia, microcephaly, contractures of hips and knees, hypoplastic scrotum, undescended testes, prominent, cup-shaped ears, narrow bifrontal diameter, broad nasal root, prominent epicanthal folds, bilateral clinodactyly V, ataxic gait, progressive joint contractures and muscle wasting of lower extremities, mixed hearing loss and hypoplastic inferior cerebellar vermis, partial dysgenesis of corpus callosum, and narrow pons and brain stem on MRIHPDeletion† 10q26.13 1920 126,415,527→ 134,032,9117,617,384D/526Page 6 of 20(page number not for citation purposes)including R3, HTT, 1, PDE6B, d WHSC1The deletion in this patient includes the Wolf- Hirschhorn syndrome critical region, and the clinical features are compatible with that syndrome [74].Pathogenicincluding RA2C, , EVCThe deletion includes the Wolf- Hirschorn syndrome critical region.Pathogenicncluding , CLN8, d MCPH1D and 5 genesDeletion is homozygous in child, heterozygous in both parents (not de novo mutation)Not pathogenic for IDincluding 0A1, FRK, , HDAC2, M9, PLN, d WISP3Pathogenicincluding DGKB, AM126A, L6, and EF5PathogenicTable 2: Genomic imbalance and uniparental disomy detected by 500K GeneChip® AGH in the new cohort. (Continued)BMC Genomics 2009, 10:526http://www.biomedcentral.com/1471-2164/106473 Deletion 4p16.3 337 190,631→ 3,277,4363,086,805 Cytogenetic re-analysis, FISH3 year-old male with fetal growth retardation, length 2 standard deviations below the mean for age, weight 3-4 standard deviations below the mean, head circumference 3-4 standard deviations below the mean, global developmental delay, seizures, triangular face, small jaw, posteriorly rotated ears, 2° hypospadias, and ataxia~45 genes ADD1, FGFIDUA, LETMSH3BP2, an5814 Deletion† 4p16.1-p16.31520 13,255→ 8,472,6578,459,402 Cytogenetic re-analysis, MLPA13 month-old female with length below the 3rd centile, microcephaly, developmental delay, bilateral preauricular pits, and submucus cleft palate~90 genes ADD1, ADCRMP1Duplication†8p23.1-p23.32579 180,568→ 6,898,0766,717,508 20 genes iARHGEF10DLGAP2, an216 Deletion 6p21.33 42 29,937,087→ 30,026,51789,430 MLPA 18 year-old male with postnatal onset growth retardation, unilateral sensorineural deafness, narrow face, bulbous nasal tip and mild intellectual disability.HCG9, MICpseudo3160 Deletion 6 q21-q22.311596 111,979,175→ 121,506,9169,527,741 FISH 8 year-old female with moderate developmental delay, hypotonia, microcephaly, brachycephaly, epicanthic folds, small ears with hypoplastic lobes, and micrognathia~40 genes ASF1A, COL1FYN, GOPCLAMA4, MCTSPYL1, an2200 Deletion 7p15.3 2320 14,141,506→ 24,950,41410,808,908 FISH 11 1/2 year-old female with head circumference at the 2nd centile, mild cognitive impairment, sensorineural hearing loss, cleft palate, craniosynostosis, unilateral ptosis and esotropia, orbital rim hypoplasia, malar and midface hypoplasia, low-set ears with incomplete out-folding of superior helix, brachydactyly and syndactyly of digits, broad thumbs, decreased range of motion in elbows, and leg length discrepancy~40 genes DFNA5, DNAH11, FHDAC9, IRAPG/526Page 7 of 20(page number not for citation purposes)th including B, CYP3A5, C3A, SMURF1, FR2Pathogenicncluding 7, RAB2A, OX, and APathogenicincluding 1, DNAJB5, LT, GBA2, R, NPR2, K, SHB, 13B, and PThe deleted region in this patient is completely included in the region deleted in patient 9346.Pathogenicincluding 1, DNAJB5, LT, GBA2, R, NPR2, K, SHB, 13B, and PThe deleted region in this patient includes the entire segment deleted in Patient 663.Pathogeniccluding T1This deletion is within the critical region for the 9q subtelomeric deletion syndrome[75], and the child's clinical features are compatible with that syndrome.PathogenicTable 2: Genomic imbalance and uniparental disomy detected by 500K GeneChip® AGH in the new cohort. (Continued)BMC Genomics 2009, 10:526http://www.biomedcentral.com/1471-2164/109938 Deletion 7q22.1 170 98,211,585→ 100,553,7552,342,170 FISH 14 year-old female with height < 5centile, weight < 5th centile, head circumference < 5th centile, severe cognitive impairment, left sensorineural hearing loss, close-set eyes, broad nasal root, marked retrognathia, high-arched palate, small and narrow feet, short 2-5th toes with hypoplastic nails, atrio-vetricular septal defect, and polyarticular arthritis~70 genes ACHE, ACTL6EPO, MUSERPINE1, and T1594 Duplication8 q12 1220 58,388,614→ 65,306,0976,917,483 FISH 1 1/2 year-old female with height > 97th centile, head circumference at 2nd-5th centile, developmental delay, Duane anomaly, broad glabella, epicanthic folds with telecanthus, upslanting palpebral fissures, pre-auricular pits, large ears, atrial and ventricular septal defects, and renal reflux15 genes iASPH, CHDRLBP1L1, TTTP663 Deletion 9 p13.3 800 34,144,847→ 38,736,4514,591,604 FISH 5 1/2 year-old female with height at the 5th centile, developmental delay, tremor, ocular hypertelorism, epicathal folds, double hair whorl, bilateral ptosis, short upturned nose, flattened philtrum, underdeveloped genitalia, and pigmentary retinal changes~75 genes CNTFR, DNAIFANCG, GAGNE, GRHPPAX5, RECTPM2, UNCVC9346 Deletion 9p11.2-p13.3880 33,702,471→ 44,744,67511,042,204 FISH 9 1/2 year-old female with moderate cognitive impairment, seizures, tremor, cataract, broad frontal area with bossing, arched eyebrows, low nasal bridge, and short, upturned nose~85 genes CNTFR, DNAIFANCG, GAGNE, GRHPPAX5, RECTPM2, UNCVC523 Deletion 9q34.3 36 139,516,033→ 139,814,485298,452 FISH 4 year-old female with moderate developmental delay, hypotonia, microcephaly, flat face with upslanting palpebral fissures, ocular hypotelorism, synophrys, and anteverted nares7 genes inEHM/526Page 8 of 20(page number not for citation purposes)rous Clinical features compatible with mosaic trisomy 9 syndrome[76,77]Pathogenicrous Mosaic paternal isodisomy; phenotype not compatible with Beckwith-Wiedemann syndromeUncertainding IRS2, MYO16Pathogenic T-cell a-chain V, n genesHighly polymorphic regionNot pathogenic for IDrous Uncertainding MAPT The deleted segment includes the critical region for the 17q21.31 deletion syndrome, [78] and this patient's clinical features are compatible with that syndrome.PathogenicTable 2: Genomic imbalance and uniparental disomy detected by 500K GeneChip® AGH in the new cohort. (Continued)BMC Genomics 2009, 10:526http://www.biomedcentral.com/1471-2164/108056 Mosaic Trisomy9 27,641 whole chromosome FISH 2 1/2 year-old male with weight < 5th centile, developmental delay, preauricular skin tags, hypospadias, and cryptorchidismNume6904 Uniparental Disomy11p11.2-pterSee Table 3 196,767→ 44,589,53044,392,763 Micro-satellite markers11 year-old female with height < 5th centile, gross and fine motor delay, hypotonia, and moderate mental handicapNume9897 Deletion 13q33.3-q34530 107,271,189→ 109,368,9962,097,807 FISH 10 year-old female with fetal growth retardation, moderate cognitive impairment, upslanting palpebral fissures, and retrognathia6 genes incluLIG4, and 818 Deletion 14q11.2 24 21,929,710→ >22,036,502106,792 Fosmid FISH (variable)§6 1/2 year-old male with weight < 5th centile, height at 5th centile, mild cognitive impairment with particular delay in language, mild mid-face hypoplasia with narrow high-arched palate, mild micrognathia, pre-auricular pit, joint laxity, bilateral clinodactyly of hands, and bilateral 2-3 syndactyly of toesMultiplereceptor alphJ, and regio1658 Uniparental Disomy16 See Table 3 Whole chromosomeWhole chromosomeMicro-satellite markers5 year-old female with normal growth, severe mental handicap, seizures, self-abusive behaviour, deep and dark creases under the eyes, mild mid-face hypoplasia, and large mouthNume2106 Deletion 17q21.31 149 41,049,321→ 41,564,451515,130 FISH 15 year-old male with fetal growth retardation, mild cognitive impairment, attention deficit disorder, sagittal craniosynostosis, long face with malar hypoplasia and mild rectrognathia, short and upslanting palpebral fissures, low-set ears, unilateral cryptorchidism, partial agenesis of the corpus callosum, and dilatation of the aortic root5 genes incluBMC Genomics 2009, 10:526http://www.biomedcentral.com/1471-2164/10/526Page 9 of 20(page number not for citation purposes)s including SYNJ1 No polymorphisms of region reportedUncertaines including BCR, 8, HIRA, MAPK1, DH, SNAP29, , SERPIND1, and TBX1The deleted segment is included in the 22q11 deletion syndrome critical region, and the phenotype is compatible with other reported cases of distal 22q11.2 microdeletion [79-81]Pathogenics including PI4KA PIND1, LZTR1, SNAP29Polymorphic region Uncertain~60 genesincludingB7, DLG3, EDA, 1, GJB1, IGBP1, 2RG, OPHN1P1L2, NLGN3, , SLC16A2, TAF1, d ZDHHC15Pathogenic children with idiopathic ID. Breakpoints are shown on n of the deletion detected by AGH. This was interpreted 8619 Deletion 21q22.11 13 33,902,218→ 34,087,893185,675 Agilent 244 K AGH24 year-old male with prenatal and postnatal growth retardation, moderate to severe intellectual disability, severe hypotonia, microcephaly, metopic craniosynostosis, cleft palate, down-slanting palpebral fissures, low-set ears, wide nasal base, retrognathia, tetralogy of Fallot, cryptorchidism, and joint hyperextensibility5 geneDeletion 22q11.2 110 19,062,809→ 19,785,125722,316 FISH 14 genDGCRPROSEPT59979 Duplication22q11.21 57 19,429,297→ 19,791,607362,310 FISH 20 year-old female with short stature, mild mental deficiency, cleft palate, and micrognathia9 geneSER1128 DuplicationXq12-q21.1475 67,088,023→ 76,204,3449,116,321 FISH 11 year-old male with normal growth, severe developmental delay, hypotonia, brachycephaly, bilateral epicanthic folds, and posteriorly rotated ears with hypoplastic helicesABCEFNBILNAPHKA1anThe table includes all de novo CNVs, mosaic trisomy and UPD detected by 500 K AGH and confirmed by an independent method in 100Human Genome Assembly Build 36.1.* The approximate number of RefSeq genes for each CNV is given, but only the most likely genes for the phenotype are named.† Unbalanced reciprocal translocation.§ Interphase FISH in patient 818 showed some cells with no signals, some with 1 signal and some with two signals for a probe in the regioas evidence of somatic mosaicism for this deletion.Table 2: Genomic imbalance and uniparental disomy detected by 500K GeneChip® AGH in the new cohort. (Continued)BMC Genomics 2009, 10:526 http://www.biomedcentral.com/1471-2164/10/526Page 10 of 20(page number not for citation purposes)De novo CNVs detected with 500 K but not 100 K AGH in children with idiopathic IDFigure 1De novo CNVs detected with 500 K but not 100 K AGH in children with idiopathic ID. The plots show in silico com-parison of estimated copy number in child versus mother (left) and child versus father (right) at each position along the chro-mosome. Upper panel: Smoothed copy number plots for chromosome 8 in Family 3890. Affymetrix 100 K AGH is shown with a 59 SNP window, and Affymetrix 500 K AGH is shown with a 170 SNP window. Note duplication at 111,442,951 to 113,003,770 bp that is apparent on 500 K AGH but was not called on our original analysis of the 100 K AGH data. The CNV is represented by 59 SNPs on the 100 K array. Lower panel: Smoothed copy number plot for chromosome 4 in Family 4840. Affymetrix 100 K AGH is shown with a 13 SNP window, and Affymetrix 500 K AGH is shown with a 145 SNP window. Note deletion at 1,346,924 to 2,846,261 bp that is apparent on 500 K AGH but was not called as de novo by 100 K AGH on our ini-tial analysis. This CNV is represented by 17 SNPs on the 100 K array.BMC Genomics 2009, 10:526 http://www.biomedcentral.com/1471-2164/10/526have occurred by chance because we just happened toinclude a few more patients with such genomic changes inthe new cohort than in the 100 K cohort, we also foundtwo additional de novo CNVs by 500 K GeneChip® AGHWe detected three apparently de novo CNVs smaller than200 kb among the 100 trios tested with 500 K GeneChip®AGH in the new cohort (a 107 kb deletion of chromo-some 14 in Patient 818, a 186 kb deletion of chromosomeTable 3: Genomic imbalance detected by 500K AGH in 54 idiopathic ID trios from the 100K cohort.Affymetrix 100K AGH Affymetrix 500K AGHFamily Change Location Start → End Size (bp) Change Location Start → End Size (bp)1895 Deletion 13q12.11-q12.13 18,867,056→ 24,517,7305,650,674 Deletion 13q12.11-q12.12 18,876,037→ 24,330,2325,454,1953476 Deletion 4q21.21-q22.1 82,008,594 → 93,076,27811,067,684 Deletion 4q21.21-q22.1 82,429,950→ 91,434,3379,004,3873890 Normal Duplication 8q23.2-q23.3 111,442,951→ 113,003,7701,560,8194794 Duplication 16p13.3 925,718→ 3,864,9382,939,220 Duplication 16p13.3 2,681,813→ 3,927,5241,245,7114818 Deletion 12q14.2-q15 63,342,649→ 66,780,0953,437,446 Deletion 12q14.2-q15 63,362,084→ 66,737,6993,375,6154840 Normal Deletion 4p16.3 1,346,924→ 2,846,2611,499,3375003 Deletion 2p16.3 50,799,281→ >51,120,644321,363 Deletion 2p16.3 50,829,675→ 51,120,302290,6275566 Deletion 14q11.2 20,741,117→ 20,918,741177,624 Deletion 14q11.2 20,787,740→ 20,988,716200,9765994 Mosaic Trisomy 9 Whole Chromosome Mosaic Trisomy 9 Whole Chromosome6545 Deletion 7p22.2-p22.1 3,498,135→ 7,134,2183,636,083 Deletion 7p22.2-p22.1 3,657,805→ 6,165,5972,507,7927807 Deletion 22q12.1 26,144,210→ 27,557,9711,413,761 Deletion 22q12.1 26,293,416→ 27,462,4581,169,0428326 Deletion 14q11.2 19,584,863→ 21,207,9351,623,072 Deletion 14q11.2 19,592,409→ 21,256,8221,664,413The table includes all de novo CNVs and mosaic trisomy detected by 500 K AGH and confirmed by an independent method in a selected group of 54 trios who had previously been tested by 100 K GeneChip® AGH [10]. Breakpoints are shown on Human Genome Assembly Build 36.1.Page 11 of 20(page number not for citation purposes)among the 44 children whose 100 K AGH was interpretedas normal in our earlier studies (Figure 1 and Table 3).21 in Patient 8619, and an 89 kb deletion of chromosome6 in Patient 216 that was actually a homozygous lossinherited from two heterozygous parents), but none ofBMC Genomics 2009, 10:526 http://www.biomedcentral.com/1471-2164/10/526these CNVs was clearly pathogenic. The overall size distri-bution of pathogenic CNVs detected by 500 K GeneChip®AGH among 154 children with idiopathic ID in thepresent study is similar to that observed by 100 K Gene-Chip® AGH among 100 children with idiopathic ID whomwe studied previously [10] (see Additional File 2: Supple-mental Figure S1). The higher detection rate on the 500 Karray therefore appears to be related more to better probecoverage in relevant genomic regions and an improvedability to distinguish CNVs from background noise, ratherthan to a capacity to identify much smaller pathogenica 1.5 Mb deletion of 4p16.3, respectively, are obvious onthe 500 K AGH but were not called on the 100 K analysis.In retrospect, the 4p16.3 deletion in Patient 4840 can beseen on the 100 K AGH copy number plot despite thenoisy data, but it was not called by either the automatedanalysis or visual inspection of these plots when the initialstudy was done. Our failure to detect the de novo duplica-tion of 8q23.2q23.3 in Patient 3890 was probably causedby the noisy data in the father's study.Distinguishing benign CNVs from those that cause ID andUniparental disomy detected with Affymetrix 500 K AGH in two patients with idiopathic IDFigure 2Uniparental disomy detected with Affymetrix 500 K AGH in two patients with idiopathic ID. A) The child in Family 6904 was found to have mosaic paternal uniparental disomy, probably isodisomy, of chromosome 11 p15.5-p11.2. SNP genotypes obtained by Affymetrix 500 K AGH and interpreted for the trio as described in the Methods are shown along the length of chro-mosome 11. B) The child in Family 1658 was found to have maternal uniparental disomy for all of chromosome 16. The ends of both chromosome arms (proximal to 11,559,620 bp and distal to 84,641,383 bp) appear to be isodisomic; the central portion of the chromosome is heterodisomic. SNP genotypes obtained by Affymetrix 500 K AGH and interpreted for the trio as described in the Methods are shown along the length of chromosome 16. C) Allelic imbalance, compatible with paternal isodisomy and mosaicism, for two informative microsatellite markers in the involved region of chromosome 11 in the child in Family 6904. The location of each marker is shown in brackets. D) Maternal heterodisomy for two informative microsatellite  markers in the involved region of chromosome 16 in the child in Family 1658. The location of each marker is shown in brackets.Page 12 of 20(page number not for citation purposes)CNVs. This is illustrated in Families 3890 and 4840 (Fig-ure 1), in which a 1.6 Mb duplication of 8q23.2q23.3 andother birth defects is a critical issue in routine clinical useof AGH. Benign CNVs occur in all people and are a majorBMC Genomics 2009, 10:526 http://www.biomedcentral.com/1471-2164/10/526source of genetic variation in the normal population[21,27,29]. Most apparently benign CNVs over 2 kb insize occur as polymorphisms with minor allele frequen-cies of at least 5% [21] and are inherited from a parent[21,23,45,46].Benign and pathogenic CNVs can usually be distinguishedin patients with ID and other birth defects by inheritanceand genotype-phenotype correlation [5,33,47]. In thisstudy, we identified a mean of about 10 CNVs per subjectin the 154 ID trios tested by 500 K AGH. The vast majorityof these CNVs were characterized as benign because theywere inherited from a normal parent. Genomic imbalancethat occurs de novo in a patient with ID whose parents arenormal is more likely to be pathogenic than genomicimbalance that was inherited unchanged from a normalparent. We performed AGH on both parents of every childwith ID to determine the inheritance of the CNVs foundin the child, but this is sometimes not possible in clinicalpractice. In such instances it is necessary to infer likely denovo occurrence by information obtained from popula-tions that have previously been studied [19,22-24,48,49].Great care must be taken to avoid misinterpretation whenthis is done, especially if the available data were obtainedwith lower resolution AGH, the phenotypic characteristicsof the comparison population are uncertain, reported pol-ymorphic CNVs do not have exactly the same breakpointsas the CNV of interest, or the population frequency of apreviously-reported CNV is unknown.Compelling evidence that a CNV in a person with ID ispathogenic exists if the genomic imbalance is known tocause the patient's phenotype in other individuals, e.g., ifdrome (Patient 9133), or a child with del 17q21.31 hasfeatures of the syndrome associated with this deletion(Patient 2106). Pathogenicity is also supported when aCNV includes a gene that is known to cause the patient'sphenotype when inactivated (if the CNV is a deletion) orover-expressed (if the CNV is a duplication). On the otherhand, a CNV is unlikely to be pathogenic if it involves ahighly polymorphic region in which genomic loss (orgain, whichever is present in the patient) of the entireinvolved segment is known to occur in normal people.If a direct genotype-phenotype correlation of this kindcannot be made in a particular case, certain genetic fea-tures of the CNV may provide clues to its pathogenicity.CNVs that are larger and those that involve gene-richregions are more likely to be pathogenic than CNVs thatare smaller and involve only gene-poor regions [5,47]. Inaddition, clinical experience suggests that deletions aremore likely to be pathogenic than duplications [47]. Thegenetic content of a CNV may also make pathogenicitymore or less likely. For example, involvement of a genethat lies within a pathway that is known to contain otherdosage-sensitive genes associated with a similar pheno-type strengthens the possibility of pathogenicity, while aCNV that does not contain any genes that are expressed inrelevant tissues during embryogenesis is unlikely to bepathogenic for ID.There are, of course, exceptions to each of these "rules".Some benign CNVs arise de novo [21,23,45,46,50], asappears to have occurred in the de novo deletion of chro-mosome 14q11.2 we found in Patient 818. The 107 kbregion involved is highly polymorphic and contains sev-Table 4: UPD detected in 100 children with idiopathic ID from the new cohort.Patient Chromosome Affected Region SNPs in Region of UPD InterpretationStart End Size (bp) Total Paternal UPD Maternal UPD BPI* MI*6904 11 196,767 44,589,530 44,392,763 9,857 h = 192*i = 456*h = 89i = 159 8 Paternal Isodisomy1658 16 14,139 11,559,620 11,545,481 2,837 h = 0i = 1h = 193i = 1885 19 Maternal Isodisomy11,559,620 84,641,380 73,081,760 11,698 h = 0i = 0h = 638i = 312 95 Maternal Heterodisomy84,641,383 88,668,856 4,027,473 772 h = 0i = 0h = 25i = 561 7 Maternal IsodisomyThe table includes all UPD detected by 500 K GeneChip® AGH and confirmed by an independent method in 100 children with idiopathic ID. All mendelian inconsistencies observed were single. SNPs that are not listed as paternal UPD, maternal UPD, BPI* or MI* were uninformative with respect to UPD. Breakpoints are shown on Human Genome Assembly Build 36.1.* Abbreviations used: h = heterodisomy, i = isodisomy, BPI = biparental inheritance, MI = mendelian inconsistency.Page 13 of 20(page number not for citation purposes)a child with del 9q34.3 has features of the 9q subtelom-eric deletion syndrome (Patient 523), a child with del1p36.32p36.33 has features of the 1p36 deletion syn-eral T-cell receptor variable region genes. On the otherhand, some CNVs that are inherited from a normal parentare pathogenic for ID. Examples include maternal trans-BMC Genomics 2009, 10:526 http://www.biomedcentral.com/1471-2164/10/526mission of a UBE3A deletion to a child with Angelmansyndrome [51], maternal transmission of a MECP2 dupli-cation to a son [52], and CNVs such as dup 22q11.2[53,54] or del 1q21.2 [55] that can cause ID but exhibitincomplete penetrance.Although large (> 250 kb) CNVs are often pathogenic,they may be benign [19,29]. Most benign CNVs are small(< 250 kb) [19,27,29], and it seems probable that thesmaller a CNV, the more likely it is to be benign. Never-theless, no clear size distinction exists between benignand pathogenic CNVs. We found pathogenic CNVs assmall as the 298 kb deletion of 9q34.3 in Patient 523 inthis study, and others have reported even smaller patho-genic CNVs [13-15,56-58]. Expression patterns, func-tional annotation and animal models can provideimportant clues to pathogenicity in some cases, but with-out knowledge of the phenotypic effects of a copy numberalteration in humans, one can rarely, if ever, be certainwhether a novel gain or loss of a particular genomicregion can produce ID or other birth defects.In our 500 K AGH study of 154 ID trios, 58 de novo CNVswere called by bioinformatic analysis, and 33 of theseCNVs were confirmed and shown to be de novo by an inde-pendent method. Because we could assess the phenotypesof our patients in detail and correlate the findings withthose obtained by AGH, we were able to determine withconfidence whether the genomic imbalance we observedwas pathogenic or not in every case studied except three(Tables 2 and 3). Such genotype-phenotype correlation iscritical to determining the effects of novel CNVs detectedby AGH in patients with ID.A CNV of uncertain clinical significance was encounteredin three (1.9%) of the 154 trios analyzed in this series - a362 kb duplication of 22q11.21 in Patient 9979 (Table 2),a 186 kb deletion of 21q22.11 in Patient 8619 (Table 2)and a 1.6 Mb duplication of chromosome 8q23.2q23.3 inPatient 3890 (Table 3). This rate of CNVs of uncertainclinical significance is similar to that reported in largeseries of patients with ID and other birth defects studiedby AGH with "targeted" chips [31,35,59].We were uncertain of the clinical significance of eithercase of UPD that we detected. Although only a few live-born children with UPD 16 have been recognized, thereported experience does not suggest that UPD 16 cancause the abnormalities observed in Patient 1658 [60,61].Paternal UPD 11p15 can produce Beckwith-Wiedemannsyndrome [62], but this phenotype is very different fromthat observed in the affected child in Family 6904. How-ever, as both of these cases involved isodisomy of a por-Although the detection of UPD in addition to alterationsof copy number is a theoretical benefit of using an arraythat includes probes for SNPs, the clinical utility ofgenome-wide screening for UPD in patients with idio-pathic ID and other birth defects is uncertain.We detected more pathogenic CNVs with 500 K AGH thanwith 100 K AGH, but some CNVs that were present amongour patients were not detected using the 500 K assay. Forexample, our 500 K GeneChip® analysis failed to identifya pathogenic 83 kb deletion of chromosome 16p13.3(3,862,993 bp to 3,945,522 bp) involving the CREBBPgene (Patient 5121). This de novo deletion was found byAGH on the Agilent® 244 K platform and was confirmedby MLPA. The patient is an 8 year-old boy whose clinicalfeatures are characteristic of the Rubinstein-Taybi syn-drome, which has been associated with deletions andother mutations of CREBBP in other patients [65,66]. The83 kb genomic region deleted in our patient is poorly rep-resented on the 500 K GeneChip® arrays, with a total ofonly 15 SNPs. SNP arrays have uneven genomic coverage,and the addition of non-polymorphic oligonucleotideprobes to the design of arrays like the one used in thisstudy has been shown to provide substantially betterdetection of CNVs [21].ConclusionAffymetrix GeneChip® 500 K array genomic hybridizationperformed in individuals with idiopathic intellectual dis-ability detected pathogenic genomic imbalance in 10 of10 patients in whom 100 K GeneChip® array genomichybridization found genomic imbalance, 1 of 44 patientsin whom 100 K GeneChip® array genomic hybridizationhad found no abnormality, and 16 of 100 patients whohad not previously been tested. Further improvements inarray design, ongoing improvements in AGH software,and continuing enhancement of resources like DECI-PHER https://decipher.sanger.ac.uk/ and the TorontoDatabase of Genomic Variants http://projects.tcag.ca/variation/ are helping to establish AGH as the primary clini-cal tool for recognition of genomic imbalance that causesID and other birth defects [33,34,37,41,67-70]. It seemslikely, however, that no perfect AGH platform for detec-tion of pathogenic CNVs may ever exist and that effectiveclinical interpretation of these studies will continue torequire considerable skill and experience [33,50,71].MethodsPatients and FamiliesWe studied 100 children with idiopathic ID who had notbeen studied by AGH before ("the new cohort") and 54 ofthe idiopathic ID patients whom we had previously testedwith 100 K Affymetrix GeneChip® AGH ("the 100 KPage 14 of 20(page number not for citation purposes)tion of the chromosome, we cannot rule out thepossibility that the abnormal phenotype was produced byhomozygosity for a recessive mutant allele [63,64].cohort"). Ten of the 54 patients in the 100 K cohort hadpreviously been found to have pathogenic genomicimbalance; the other 44 patients had previously beenBMC Genomics 2009, 10:526 http://www.biomedcentral.com/1471-2164/10/526reported to have normal 100 K GeneChip® AGH (seeAdditional File 1: Supplemental Table S1) [10,16]. Wealso performed 500 K AGH on both unaffected parents ofeach child.All of the children were assessed by a clinical geneticistwho was unable to determine the cause of the ID despitethorough clinical evaluation and testing that includedroutine karyotyping with at least 450-band resolution.Subjects were selected for AGH testing because they hadID or developmental delay and at least one of the follow-ing additional clinical features: one major malformation,microcephaly, abnormal growth, or multiple minoranomalies. Informed consent was obtained from eachfamily, and assent was also obtained from the child, ifpossible. The study was approved by the University ofBritish Columbia Clinical Research Ethics Board.DNA PreparationDNA was extracted from whole blood with a Gentra Pure-gene DNA Purification Kit (Qiagen, Hilden, Germany)according to the manufacturer's instructions. The DNAwas precipitated in 70% alcohol, resuspended in hydra-tion solution, and stored at 4°C.Hybridization to GeneChip® Mapping 500 K ArraysDNA degradation, labeling, hybridization, and scanningwere performed according to the manufacturer's protocolshttp://www.affymetrix.com using an Affymetrix FluidicsStation 450, Affymetrix Hybridization Oven 640 andAffymetrix GeneChip Scanner 3000 (Affymetrix, Inc.,Santa Clara, CA, USA).Copy Number AnalysisChip-to-chip normalization, standardization to a refer-ence, genotype detection, and copy number estimation ona single SNP basis were performed using the AffymetrixPower Tools (version 1.6.0) software suite http://www.affymetrix.com, as previously described [30]. Esti-mation of CNV boundary positions was done using Sig-nificance of Mean Difference (SMD), a method that wedeveloped. Briefly, the mean of SNP copy number esti-mates (or log2 ratios) within a CNV was compared to themean of those on the rest of the chromosome, and theprobability that the null hypothesis of Student's t-test wastrue, i.e., that the means were from the same distribution,was calculated. A search using this statistic was conductedover different CNV lengths to find the position and length(in number of SNPs) that yielded the lowest probability,defining the boundaries of a putative CNV. For each sam-ple, a random data set was produced by shuffling thegenomic positions of the data, and an identical search wasconducted. The results of this search represent false dis-The p-value distribution of apparent CNVs detected bySMD in all 462 samples (both normal parents and theaffected child from 154 ID trios) is shown in Figure 3. Weobserved that a CNV call with a p-value of 1 × 10-8 usuallyhad a false discovery rate of less than 5%, while a call witha p-value of 1 × 10-7 often had a false discovery rate of30%, with some variation from sample to sample.Since SMD compares a putative CNV to the rest of thechromosome, aberrations on the X chromosome aredetected equivalently well for males and females. Thepseudo-autosomal regions of the X-chromosomes areexceptions, but CNVs there can be detected as a functionof the reference set used in the analysis. GeneChip® 500 Karrays do not contain Y-chromosome probes.The SMD search was performed on each child with eachparent as a reference. Since our goal was to find duplica-tions and deletions that were de novo, a criterion for selec-tion of CNVs for further analysis was that the child havethe same aberration with each parent as reference. Correctparental relationships were confirmed in all trios by use ofthe SNP genotyping calls.P-value distribution of apparent CNVs detected by SMD in 462 samples from 154 ID triosFigur  3P-value distribution of apparent CNVs detected by SMD in 462 samples from 154 ID trios. Data are from the analysis performed by 500 K GeneChip® AGH. Some of these apparent CNVs were merged into larger CNVs at a later stage of the analysis, but most represent individual aber-rations. Apparent CNVs with p-values less than 1 × 10-8 were analysed further to determine if they were inherited or had occurred de novo. 4577 apparent CNVs were found with p-values below this threshold (shown in blue in the figure). The bin plotted at 10-17 actually contains all CNVs with p < 10-16, which is the lower limit of the tables used to calculate p-val-Page 15 of 20(page number not for citation purposes)coveries due to the random variation of the individualSNP data.ues.BMC Genomics 2009, 10:526 http://www.biomedcentral.com/1471-2164/10/526Every putative de novo CNV call was evaluated by anotheranalysis conducted using a large reference set of individu-als. Early in the study, the 48 sample reference set availa-ble from Affymetrix was used, but later a set of 50 mothersfrom our own data was used. The use of a local referenceset significantly reduced noise. The use of the large refer-ence set was required to detect the occurrence of aberra-tions in both parents that were not inherited by the child.Putative de novo CNVs were further evaluated by visualexamination of the copy number plots in comparison toboth parents as well as of the plots for the child and bothparents in comparison to the large reference set.Validation of De Novo CNVsPutative de novo CNVs identified by 500 K AGH were val-idated by fluorescence in situ hybridization (FISH) or mul-tiplex ligation-dependant probe amplification (MLPA),Agilent® 244 K AGH, or repeat cytogenetic analysis. FISHwas performed with BAC or fosmid probes selected usingthe University of California at Santa Cruz GenomeBrowser [72] and the May 2006 assembly of the humangenome sequence. BAC or fosmid DNA was isolated bysmall-scale (miniprep) preparation and was labeled withSpectrum Red or Green (Vysis, Abbott Molecular, AbbottPark, IL, USA) by use of a Vysis nick translation reagentkit. The labeled product was mixed with 3 mg of humanCot-1 DNA (Invitrogen, Life Technologies Corporation,Carlsbad, CA, USA) and was isolated by means of a stand-ard DNA precipitation method. Cytogenetic pellets wereprepared according to standard clinical procedures, andchromosomes and nuclei were visualized by counterstain-ing with 4',6-diamidino-2-phenylindole. For deletions, atleast 10 metaphase cells were analyzed, and interphasenuclei were examined but not counted. For duplications,at least 10 metaphase cells and at least 50 interphasenuclei were analyzed. All FISH probes were tested on met-aphase spreads from unaffected individuals to assureproper hybridization.MLPA was performed using the P070 subtelomeric kit(MRC Holland, Amsterdam, The Netherlands); all posi-tive results were confirmed with another kit (P036B). Theprocedure was conducted according to the manufacturer'srecommendations. Briefly, the patient's DNA was dilutedin PCR-grade water and quality was assessed via spectro-photometry (Nanodrop®, Thermo Scientific, Wilmington,DE, USA). The hybridization solution (SALSA probe-mixand MLPA buffer) was added to a final DNA concentra-tion of 60 ng/μl. DNA was denatured at 90°C, thenhybridized for 16-20 hours at 60°C. Ligation was per-formed at 54°C for 15 minutes, and the ligated productwas denatured at 98°C for 5 minutes and then amplifiedby PCR (SALSA PCR buffer, PCR-primers and polymer-gies, Carlsbad, CA, USA). Normalization of the data andanalysis of the MLPA results were conducted using Coffa-lyser v3.5 software provided by MRC Holland (Amster-dam, The Netherlands).AGH was performed with Agilent® 244 K oligonucleotidearrays (Agilent Technologies Inc., Santa Clara, CA, USA)according to the manufacturer's instructions. Two arrayswere used for each trio, one in which the child's DNA washybridized against the father's DNA, and another inwhich the child's DNA was hybridized against themother's DNA. Captured images were analysed with Fea-ture Extraction v 9.1 and CGH Analytics 3.5.14 (AgilentTechnologies Inc., Santa Clara, CA, USA).Cytogenetic analysis was performed on peripheral bloodcultures after preparation and G-banding of metaphasechromosomes using standard clinical methods.Uniparental Disomy (UPD)Given the genotypes for the child, mother, and father,errors in mendelian transmission were identified andtheir frequency compared to normal or technical errorrates, which were very low. Essentially, the occurrences ofan AA parent with a BB child, or vice versa, were counted.Graphical tools were developed to distinguish heterodis-omy and isodisomy by visualization and to determine theparent of origin [30].Confirmation of uniparental disomy was obtained bygenotyping microsatellite markers chosen for high hetero-zygosity values. The following markers were genotypedand were informative for chromosome 11: D11S1363(AFMA134WH5), D11S4046 (AFMB042YF5), D11S1318(AFM218XE1), D11S4088 (AFMA155TE9) andD11S4146 (AFMB072WE5). The following markers weregenotyped and were informative for chromosome 16:D16S3093 (AFMB308ZH9), D16S409 (AFM161XA1),D16S515 (AFM340YE5) and D16S402 (AFM031XA5). 25ng of DNA diluted in 10:1 TE buffer were amplified foreach PCR reaction. Primers fluorescently labeled witheither 6-fam or HEX (Applied Biosystems, Life Technolo-gies, Carlsbad, CA, USA) were used in conjunction withAmpliTaq Gold® PCR kit reagents (Applied Biosystems,Life Technologies, Carlsbad, CA, USA). PCR was per-formed using the following steps: 95°C for 10 minutes;30 cycles of 95°C for 30 seconds, 55°C for 30 seconds,and 72°C for 90 seconds; and a final step at 72°C for 7minutes. The resulting PCR product in a volume of 1 μlwas combined with 9 μl formamide (Applied Biosystems,Life Technologies, Carlsbad, CA, USA) and 0.3 ul GeneS-can ROX 500 (Applied Biosystems, Life Technologies,Carlsbad, CA, USA), denatured at 95°C for 5 minutes,Page 16 of 20(page number not for citation purposes)ase). The PCR product was run for fragment analysis on anABI 3130 sequencer (Applied Biosystems, Life Technolo-quickly chilled in ice and loaded onto a Genetic Analyzer3130XL (Applied Biosystems, Life Technologies,BMC Genomics 2009, 10:526 http://www.biomedcentral.com/1471-2164/10/526Carlsbad, CA, USA). Data were visualized using GeneMa-pper v4.0 (Applied Biosystems, Life Technologies,Carlsbad, CA, USA).List of Abbreviations UsedAGH: array genomic hybridization; BAC: bacterial artifi-cial chromosome; bp: base pairs; CNV: copy number var-iant; del: deletion; FISH: fluorescence in situhybridization; ID: intellectual disability; K: thousand; Mb:megabase; MLPA: multiplex ligation-dependant probeamplification; PCR: polymerase chain reaction; SMD: sig-nificance of mean difference; SNP: single nucleotide poly-morphism; UPD: uniparental disomy.Authors' contributionsJMF and MAM conceived, designed and coordinated thisstudy. PB developed and implemented the consent proc-ess, and JMF, SA, LA, LA, PB, CB, WTG, SL, EL, PM, JLM,BCM, MSP, GAR, MIVA and S-LY recruited patients. Clin-ical evaluations were performed by LA, LA, CB, JMF, WTG,SL, EL, PM, BCM, MSP, MIVA and S-LY. AB and SC per-formed the array genomic hybridizations, and AB, ADD,PE, SF, SL, HIL, HQ, MAM and JMF interpreted the AGHdata. Confirmatory FISH and MLPA studies were done byPE, DC, EL and JM. LA, LA, CV, PE, WTG, SL, EL, PM, JLM,BCM, MSP, MIVA, S-LY, FRZ and JMF performed the gen-otype-phenotype correlations. JMF drafted the manu-script, which was critically reviewed and edited by AB,ADD, PE, SL, JLM, MSP, FRZ and MAM. All authors readand approved the final manuscript.Additional materialAcknowledgementsThis work was supported by grants from Genome Canada (JMF, MAM),Genome British Columbia (JMF, MAM), the Canada Foundation for Innovation (JMF), British Columbia Knowledge Development Fund (JMF), Réseau de Médecine Génétique Appliquée of the Fonds de la Recherche en Santé du Québec (JLM, EL) and by the Fonds d'encouragement à la recher-che clinique du CHU Sainte-Justine (JLM, EL). The authors thank Jacquie Schein for providing the BACs used for FISH confirmation and Tracy Tucker for her helpful comments regarding the manuscript.References1. Ford C, Miller O, Polani P, de Almeida J, Briggs J: A sex-chromo-some anomaly in a case of gonadal dysgenesis (Turner's syn-drome).  Lancet 1959, 1:711-3.2. Jacobs P, Strong J: A case of human intersexuality having a pos-sible XXY sex-determining mechanism.  Nature 1959,183:302-3.3. 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The findings in families in whom de novo CNVs were found are summarized in Table 3.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-10-526-S1.DOC]Additional file 2Supplemental Figure S1. Size distribution of pathogenic CNVs detected by 100 K and 500 K AGH in idiopathic ID trios. The number of deletions (red bars) or duplications (blue bars) in each size class is shown for 8 submicroscopic deletions and 2 submicroscopic duplications found in 100 idiopathic ID trios studied with 100 K AGH (empty and stippled bars) and for 22 submicroscopic deletions and 5 submicroscopic duplications found in 154 idiopathic ID trios studied with 500 K AGH (filled bars and stippled bars). 46 trios in the 100 K cohort were studied only with 100 K GeneChip® AGH (empty bars), 54 other trios from the 100 K cohort were studied with both 100 K and 500 K GeneChip® AGH (stippled bars), and 100 additional trios constituted a new cohort who were studied only with 500 K GeneChip® AGH (solid bars). Data for the 100 K trios are from [10]. 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