Title: Oligonucleotide Microarray Analysis of Genomic Imbalance in Children with Mental Retardation
Abstract: The cause of mental retardation in one-third to one-half of all affected individuals is unknown. Microscopically detectable chromosomal abnormalities are the most frequently recognized cause, but gain or loss of chromosomal segments that are too small to be seen by conventional cytogenetic analysis has been found to be another important cause. Array-based methods offer a practical means of performing a high-resolution survey of the entire genome for submicroscopic copy-number variants. We studied 100 children with idiopathic mental retardation and normal results of standard chromosomal analysis, by use of whole-genome sampling analysis with Affymetrix GeneChip Human Mapping 100K arrays. We found de novo deletions as small as 178 kb in eight cases, de novo duplications as small as 1.1 Mb in two cases, and unsuspected mosaic trisomy 9 in another case. This technology can detect at least twice as many potentially pathogenic de novo copy-number variants as conventional cytogenetic analysis can in people with mental retardation. The cause of mental retardation in one-third to one-half of all affected individuals is unknown. Microscopically detectable chromosomal abnormalities are the most frequently recognized cause, but gain or loss of chromosomal segments that are too small to be seen by conventional cytogenetic analysis has been found to be another important cause. Array-based methods offer a practical means of performing a high-resolution survey of the entire genome for submicroscopic copy-number variants. We studied 100 children with idiopathic mental retardation and normal results of standard chromosomal analysis, by use of whole-genome sampling analysis with Affymetrix GeneChip Human Mapping 100K arrays. We found de novo deletions as small as 178 kb in eight cases, de novo duplications as small as 1.1 Mb in two cases, and unsuspected mosaic trisomy 9 in another case. This technology can detect at least twice as many potentially pathogenic de novo copy-number variants as conventional cytogenetic analysis can in people with mental retardation. Mental retardation (MR) produces life-long disability, and its burden on affected families and society is enormous. Moderate-to-severe MR, which occurs in ∼1% of the population,1Roeleveld N Zielhuis G Gabreels F The prevalence of mental retardation: a critical review of recent literature.Dev Med Child Neurol. 1997; 39: 125-132Crossref PubMed Scopus (329) Google Scholar, 2Pope A Tarlov A Disability in America: toward a national agenda for prevention. Institute of Medicine, Washington, DC1991Google Scholar is etiologically heterogeneous. Chromosomal abnormalities are the most common recognized cause, accounting for ∼10% of MR in most case series,3van Karnebeek CD Jansweijer MC Leenders AG Offringa M Hennekam RC Diagnostic investigations in individuals with mental retardation: a systematic literature review of their usefulness.Eur J Hum Genet. 2005; 13: 6-25Crossref PubMed Scopus (205) Google Scholar, 4Leonard H Wen X The epidemiology of mental retardation: challenges and opportunities in the new millennium.Ment Retard Dev Disabil Res Rev. 2002; 8: 117-134Crossref PubMed Scopus (449) Google Scholar but no etiology is recognized in at least one-third to one-half of all affected individuals. Accurate genetic counseling and prenatal diagnosis are not available for families of children with MR in whom no etiology is recognized. These children often endure a "diagnostic odyssey" of repeated testing for many different conditions, in an attempt to find the cause. Chromosomal abnormalities are usually identified by cytogenetic analysis, a microscopic method of detecting gross gain, loss, or rearrangement of genetic material in dividing cells. There have been evolutionary improvements in karyotyping since its introduction as a routine clinical service >40 years ago,5Ford CE Jones KW Polani PE de Almeida JC Briggs JH A sex-chromosome anomaly in a case of gonadal dysgenesis (Turner's syndrome).Lancet. 1959; 1: 711-713Abstract PubMed Scopus (517) Google Scholar, 6Jacobs P Strong J A case of human intersexuality having a possible XXY sex-determining mechanism.Nature. 1959; 183: 302-303Crossref PubMed Scopus (550) Google Scholar, 7Lejeune J Gautier M Turpin M É tude des chromosomes somatiques de neuf enfants mongoliens.C R Acad Sci. 1959; 248: 1721-1722Google Scholar but cytogenetic analysis has been resistant to quantum improvements and to automation, because of the requirement for tissue culture and for highly skilled technologists to analyze the microscopic images. Standard cytogenetic analysis has the advantage of surveying the entire genome for gain or loss of genetic material in a single test, but it cannot detect imbalances of genetic segments <5–10 Mb. Over the past several years, constitutional gain or loss of genomic segments containing only 1–5 Mb of DNA has been found to be another important cause of MR.8Devriendt K Vermeesch JR Chromosomal phenotypes and submicroscopic abnormalities.Hum Genomics. 2004; 1: 126-133PubMed Google Scholar These submicroscopic chromosomal alterations are usually diagnosed by locus-specific FISH,9Xu J Chen Z Advances in molecular cytogenetics for the evaluation of mental retardation.Am J Med Genet C Semin Med Genet. 2003; 117: 15-24Crossref Scopus (72) Google Scholar a test that provides much higher resolution than that of conventional cytogenetic analysis. However, locus-specific FISH is a labor-intensive microscopic technique that uses probes specifically designed for each locus (or for the relatively small number of loci) tested. FISH is, therefore, not suitable for genomewide searches for DNA copy-number changes. Better methods are needed to perform genomewide surveys for submicroscopic genomic copy-number changes in individuals with MR. Array-based methods can provide high-resolution surveys of the entire genome for submicroscopic copy-number variants (CNVs). A few small studies using these methods have found apparently pathogenic CNVs among children with MR who had normal conventional cytogenetic analyses.10Menten B Maas N Thienpont B Buysse K Vandesompele J Melotte C de Ravel T Van Vooren S Balikova I Backx L Janssens S De Paepe A De Moor B Moreau Y Marynen P Fryns JP Mortier G Devriendt K Speleman F Vermeesch JR Emerging patterns of cryptic chromosomal imbalances in patients with idiopathic mental retardation and multiple congenital anomalies: a new series of 140 patients and review of the literature.J Med Genet. 2006; 43: 625-633Crossref PubMed Scopus (334) Google Scholar, 11Tyson C Harvard C Locker R Friedman JM Langlois S Lewis ME Van Allen M Somerville M Arbour L Clarke L McGilivray B Yong SL Siegel-Bartel J Rajcan-Separovic E Submicroscopic deletions and duplications in individuals with intellectual disability detected by array-CGH.Am J Med Genet A. 2005; 139: 173-185Crossref PubMed Scopus (87) Google Scholar, 12de Vries BB Pfundt R Leisink M Koolen DA Vissers LE Janssen IM Reijmersdal S Nillesen WM Huys EH Leeuw N Smeets D Sistermans EA Feuth T van Ravenswaaij-Arts CM van Kessel AG Schoenmakers EF Brunner HG Veltman JA Diagnostic genome profiling in mental retardation.Am J Hum Genet. 2005; 77: 606-616Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar, 13Rosenberg C Knijnenburg J Bakker E Vianna-Morgante AM Sloos W Otto PA Kriek M Hansson K Krepischi-Santos AC Fiegler H Carter NP Bijlsma EK van Haeringen A Szuhai K Tanke HJ Array-CGH detection of micro rearrangements in mentally retarded individuals: clinical significance of imbalances present both in affected children and normal parents.J Med Genet. 2006; 43: 180-186Crossref PubMed Scopus (177) Google Scholar, 14Schoumans J Ruivenkamp C Holmberg E Kyllerman M Anderlid BM Nordenskjold M Detection of chromosomal imbalances in children with idiopathic mental retardation by array based comparative genomic hybridisation (array-CGH).J Med Genet. 2005; 42: 699-705Crossref PubMed Scopus (166) Google Scholar, 15Shaw-Smith C Redon R Rickman L Rio M Willatt L Fiegler H Firth H Sanlaville D Winter R Colleaux L Bobrow M Carter NP Microarray based comparative genomic hybridisation (array-CGH) detects submicroscopic chromosomal deletions and duplications in patients with learning disability/mental retardation and dysmorphic features.J Med Genet. 2004; 41: 241-248Crossref PubMed Scopus (419) Google Scholar, 16Vissers LE de Vries BB Osoegawa K Janssen IM Feuth T Choy CO Straatman H van der Vliet W Huys EH van Rijk A Smeets D van Ravenswaaij-Arts CM Knoers NV van der Burgt I de Jong PJ Brunner HG van Kessel AG Schoenmakers EF Veltman JA Array-based comparative genomic hybridization for the genomewide detection of submicroscopic chromosomal abnormalities.Am J Hum Genet. 2003; 73: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 17Miyake N Shimokawa O Harada N Sosonkina N Okubo A Kawara H Okamoto N Kurosawa K Kawame H Iwakoshi M Kosho T Fukushima Y Makita Y Yokoyama Y Yamagata T Kato M Hiraki Y Nomura M Yoshiura K Kishino T Ohta T Mizuguchi T Niikawa N Matsumoto N BAC array CGH reveals genomic aberrations in idiopathic mental retardation.Am J Med Genet A. 2006; 140: 205-211Crossref PubMed Scopus (59) Google Scholar These studies were done with arrays made with large-insert clones, usually BACs. The pathogenic submicroscopic deletions and duplications detected in these studies range in size from 0.5 to 15 Mb. However, smaller deletions and duplications can also cause MR.18Brooks EM Branda RF Nicklas JA O'Neill JP Molecular description of three macro-deletions and an Alu-Alu recombination-mediated duplication in the HPRT gene in four patients with Lesch-Nyhan disease.Mutat Res. 2001; 476: 43-54Crossref PubMed Scopus (24) Google Scholar, 19Fridman C Hosomi N Varela MC Souza AH Fukai K Koiffmann CP Angelman syndrome associated with oculocutaneous albinism due to an intragenic deletion of the P gene.Am J Med Genet A. 2003; 119: 180-183Crossref Google Scholar, 20Ren Y Saijo M Nakatsu Y Nakai H Yamaizumi M Tanaka K Three novel mutations responsible for Cockayne syndrome group A.Genes Genet Syst. 2003; 78: 93-102Crossref PubMed Scopus (33) Google Scholar, 21Ariani F Mari F Pescucci C Longo I Bruttini M Meloni I Hayek G Rocchi R Zappella M Renieri A Real-time quantitative PCR as a routine method for screening large rearrangements in Rett syndrome: report of one case of MECP2 deletion and one case of MECP2 duplication.Hum Mutat. 2004; 24: 172-177Crossref PubMed Scopus (86) Google Scholar, 22Kinning E Tufarelli C Winship WS Aldred MA Trembath RC Genomic duplication in Dyggve Melchior Clausen syndrome, a novel mutation mechanism in an autosomal recessive disorder.J Med Genet. 2005; 42: e70Crossref PubMed Scopus (14) Google Scholar, 23Zanni G Saillour Y Nagara M Billuart P Castelnau L Moraine C Faivre L Bertini E Durr A Guichet A Rodriguez D des Portes V Beldjord C Chelly J Oligophrenin 1 mutations frequently cause X-linked mental retardation with cerebellar hypoplasia.Neurology. 2005; 65: 1364-1369Crossref PubMed Scopus (68) Google Scholar, 24Vissers LE Veltman JA van Kessel AG Brunner HG Identification of disease genes by whole genome CGH arrays.Hum Mol Genet. 2005; 14: R215-R223Crossref PubMed Scopus (125) Google Scholar The ideal technique would, therefore, identify CNVs with an even greater genomewide resolution. High-density whole-genome SNP arrays have been widely used for genotyping25Stoughton RB Applications of DNA microarrays in biology.Annu Rev Biochem. 2005; 74: 53-82Crossref PubMed Scopus (299) Google Scholar and can also be used to measure genomic copy number.26Rauch A Ruschendorf F Huang J Trautmann U Becker C Thiel C Jones KW Reis A Nurnberg P Molecular karyotyping using an SNP array for genomewide genotyping.J Med Genet. 2004; 41: 916-922Crossref PubMed Scopus (105) Google Scholar, 27Bignell GR Huang J Greshock J Watt S Butler A West S Grigorova M Jones KW Wei W Stratton MR Futreal PA Weber B Shapero MH Wooster R High-resolution analysis of DNA copy number using oligonucleotide microarrays.Genome Res. 2004; 14: 287-295Crossref PubMed Scopus (301) Google Scholar Recent studies have shown that whole-genome sampling analysis (WGSA)28Kennedy GC Matsuzaki H Dong S Liu WM Huang J Liu G Su X Cao M Chen W Zhang J Liu W Yang G Di X Ryder T He Z Surti U Phillips MS Boyce-Jacino MT Fodor SP Jones KW Large-scale genotyping of complex DNA.Nat Biotechnol. 2003; 21: 1233-1237Crossref PubMed Scopus (444) Google Scholar with Affymetrix GeneChip Human Mapping 100K array sets can identify submicroscopic CNVs as well as uniparental disomy (UPD) without copy-number change.29Bruce S Leinonen R Lindgren CM Kivinen K Dahlman-Wright K Lipsanen-Nyman M Hannula-Jouppi K Kere J Global analysis of uniparental disomy using high density genotyping arrays.J Med Genet. 2005; 42: 847-851Crossref PubMed Scopus (35) Google Scholar, 30Altug-Teber O Dufke A Poths S Mau-Holzmann UA Bastepe M Colleaux L Cormier-Daire V Eggermann T Gillessen-Kaesbach G Bonin M Riess O A rapid microarray based whole genome analysis for detection of uniparental disomy.Hum Mutat. 2005; 26: 153-159Crossref PubMed Scopus (50) Google Scholar, 31Slater HR Bailey DK Ren H Cao M Bell K Nasioulas S Henke R Choo KH Kennedy GC High-resolution identification of chromosomal abnormalities using oligonucleotide arrays containing 116,204 SNPs.Am J Hum Genet. 2005; 77: 709-726Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar We studied 100 children with idiopathic MR and their parents, using WGSA with Mapping 100K arrays to look for potentially pathogenic submicroscopic genomic changes. We studied 100 children with idiopathic MR and both of their unaffected parents, eight unaffected siblings within these families (as negative controls), and eight trios in which the child had MR and a previously recognized chromosomal abnormality or UPD (as positive controls). Each of the children with idiopathic MR was assessed by a clinical geneticist who was unable to determine the cause of the child's MR despite thorough clinical evaluation and clinical testing that included routine karyotyping with at least 450-band resolution. The children were selected because they had moderate-to-severe MR or developmental delay with at least one of the following additional clinical features: one major malformation, microcephaly, abnormal growth, or multiple minor anomalies. Informed consent was obtained from each family, and assent was also obtained from the child, if possible. The study was approved by the University of British Columbia Clinical Research Ethics Board. DNA was extracted from whole blood by use of a Gentra Puregene DNA Purification Kit by following the manufacturer's instructions. The DNA was precipitated in 70% alcohol, was resuspended in hydration solution, and was stored at 4ºC. Genomic DNA sample quality was assessed by electrophoresis in a 0.7% agarose gel, followed by SYBR Green staining and visualization by use of a Typhoon 9400 variable mode imager. DNA concentration was measured with a Bio-Tek PowerWave X spectrophotometer. A sample of 500 ng of DNA was processed according to the instructions provided in the Affymetrix GeneChip Human Mapping 100K Assay Manual.31Slater HR Bailey DK Ren H Cao M Bell K Nasioulas S Henke R Choo KH Kennedy GC High-resolution identification of chromosomal abnormalities using oligonucleotide arrays containing 116,204 SNPs.Am J Hum Genet. 2005; 77: 709-726Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar In brief, 250 ng of high-quality genomic DNA was digested with XbaI or HindIII and was ligated to XbaI or HindIII adaptors. Adaptor-ligated restriction fragments were amplified by PCR, and the purified PCR products were quantified with a Bio-Tek PowerWave X spectrophotometer. Random fragmentation and labeling were performed as described elsewhere.31Slater HR Bailey DK Ren H Cao M Bell K Nasioulas S Henke R Choo KH Kennedy GC High-resolution identification of chromosomal abnormalities using oligonucleotide arrays containing 116,204 SNPs.Am J Hum Genet. 2005; 77: 709-726Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar Samples were hybridized to GeneChip Human Mapping 50K Xba240 or Hind240 arrays in an Affymetrix Hybridization Oven 640. Washes and staining of the arrays were performed with an Affymetrix Fluidics Station 450, and images were obtained using an Affymetrix GeneChip Scanner 3000. The protocol used to identify CNVs is summarized in figure 1. Initial analysis and quality assessment of the array data were performed using GeneChip DNA Analysis Software (GDAS) version 3.0. Detection of CNVs within trios consisting of an affected child and both unaffected parents was performed using Copy Number Analyser for GeneChip (CNAG)32Nannya Y Sanada M Nakazaki K Hosoya N Wang L Hangaishi A Kurokawa M Chiba S Bailey DK Kennedy GC Ogawa S A robust algorithm for copy number detection using high-density oligonucleotide single nucleotide polymorphism genotyping arrays.Cancer Res. 2005; 65: 6071-6079Crossref PubMed Scopus (556) Google Scholar version 1.0. For each trio, three comparisons of SNP copy number were made: child versus father (as reference), child versus mother (as reference), and father versus mother (as reference). Regions of copy-number gain or loss in these comparisons were determined using the hidden Markov model output of CNAG. De novo and inherited deletions and duplications in the children were called using the rules described in table 1.Table 1CNV Detection with CNAG in TriosLineChild vs. FatherChild vs. MotherFather vs. MotherCNV in ChildTypeComment(s)1LossLossLossDeletionDe novoThe mother may have a duplication that was not inherited by the child.2LossLossNo changeDeletionDe novoAlternatively, both parents might have a duplication that was not inherited by the child.3LossLossGainDeletionDe novoThe father may have a duplication that was not inherited by the child.4LossNo changeGainDeletionInherited from motherAlternatively, the father may have a duplication that was not inherited by the child.5LossGainGainNone…The father may have a duplication and the mother may have a deletion, but the child has a normal disomic copy number. Both of the child's copies may be on one chromosome.6No changeLossLossDeletionInherited from fatherAlternatively, the mother may have a duplication that was not inherited by the child.7No changeNo changeNo changeNone…Alternatively, the child and both parents may all have the same deletion or duplication.8No changeGainGainDuplicationInherited from fatherAlternatively, the mother may have a deletion that was not inherited by the child.9GainLossLossNone…The father may have a deletion and the mother may have a duplication, but the child has a normal disomic copy number. Both of the child's copies may be on one chromosome.10GainNo changeLossDuplicationInherited from motherAlternatively, the father may have a deletion that was not inherited by child.11GainGainLossDuplicationDe novoThe father may have a deletion that was not inherited by the child.12GainGainNo changeDuplicationDe novoAlternatively, both parents may have a deletion that was not inherited by the child.13GainGainGainDuplicationDe novoThe mother may have a deletion that was not inherited by the child.Note.—"Loss," "gain," or "no change" of copy number refers to the first individual listed in comparison with the second individual listed. Our goal in this trio analysis was to identify de novo CNVs in the affected child. If the child showed the same copy-number change (gain or loss) in comparison with both parents, the child's CNV was considered to have arisen de novo, regardless of whether the copy number in both parents was the same. Open table in a new tab Note.— "Loss," "gain," or "no change" of copy number refers to the first individual listed in comparison with the second individual listed. Our goal in this trio analysis was to identify de novo CNVs in the affected child. If the child showed the same copy-number change (gain or loss) in comparison with both parents, the child's CNV was considered to have arisen de novo, regardless of whether the copy number in both parents was the same. An analysis complementary to that described above was performed using a reference set that included all 216 unaffected parents in this study. SNP copy number was assessed in each affected child and each unaffected parent in comparison with this large reference set, by use of DNA-Chip Analyzer (dChip) software (version release November 17, 2005).33Zhao X Li C Paez JG Chin K Janne PA Chen TH Girard L Minna J Christiani D Leo C Gray JW Sellers WR Meyerson M An integrated view of copy number and allelic alterations in the cancer genome using single nucleotide polymorphism arrays.Cancer Res. 2004; 64: 3060-3071Crossref PubMed Scopus (441) Google Scholar Regions of copy-number gain or loss were detected using the hidden Markov model output of dChip. De novo and inherited deletions and duplications in the children were called as described in table 2.Table 2CNV Detection with dChip in Trios by Use of a Reference SetLineChild vs. Reference SetFather vs. Reference SetMother vs. Reference SetCNV in ChildTypeComment(s)1LossLossLossDeletionInherited (from either parent)The deletion is present in both parents, and the child could have inherited it from either parent.2LossLossNo changeDeletionInherited from father…3LossLossGainDeletionInherited from fatherThe mother has a duplication that was not inherited by the child.4LossNo changeLossDeletionInherited from mother…5LossNo changeNo changeDeletionDe novo…6LossNo changeGainDeletionDe novoThe mother has a duplication that was not inherited by the child.7LossGainLossDeletionInherited from motherThe father has a duplication that was not inherited by the child.8LossGainNo changeDeletionDe novoThe father has a duplication that was not inherited by the child.9LossGainGainDeletionDe novoBoth parents have a duplication that was not inherited by the child.10No changeLossLossNone…Both parents have a deletion that was not inherited by the child.11No changeLossNo changeNone…The father has a deletion that was not inherited by the child.12No changeLossGainNone…The father has a deletion, the mother has a duplication, but the child has a normal disomic copy number. Both of the child's copies may be on one chromosome.13No changeNo changeLossNone…The mother has a deletion that was not inherited by the child.14No changeNo changeNo changeNone……15No changeNo changeGainNone…The mother has a duplication that was not inherited by the child.16No changeGainLossNone…The father has a duplication, the mother has a deletion, but the child has a normal disomic copy number. Both of the child's copies may be on one chromosome.17No changeGainNo changeNone…The father has a duplication that was not inherited by the child.18No changeGainGainNone…Both parents have a duplication that was not inherited by the child.19GainLossLossDuplicationDe novoBoth parents have a deletion that was not inherited by the child.20GainLossNo changeDuplicationDe novoThe father has a deletion that was not inherited by the child.21GainLossGainDuplicationInherited from motherThe father has a deletion that was not inherited by the child.22GainNo changeLossDuplicationDe novoThe mother has a deletion that was not inherited by the child.23GainNo changeNo changeDuplicationDe novo…24GainNo changeGainDuplicationInherited from mother…25GainGainLossDuplicationInherited from fatherThe mother has a deletion that was not inherited by the child.26GainGainNo changeDuplicationInherited from father…27GainGainGainDuplicationInherited (from either parent)The duplication is present in both parents, and the child could have inherited it from either parent.Note.—The reference set comprised the 216 unaffected parents of all the studied children with idiopathic MR, known chromosomal abnormalities, or known UPD. "Loss," "gain," or "no change" of copy number refers to the individual listed in comparison with the large reference set. Our goal in this comparison was to identify de novo CNVs in the affected child. If the child showed a copy-number change (gain or loss) in comparison with the large reference set that was not present in either parent, the child's CNV was considered to have arisen de novo. Open table in a new tab Note.— The reference set comprised the 216 unaffected parents of all the studied children with idiopathic MR, known chromosomal abnormalities, or known UPD. "Loss," "gain," or "no change" of copy number refers to the individual listed in comparison with the large reference set. Our goal in this comparison was to identify de novo CNVs in the affected child. If the child showed a copy-number change (gain or loss) in comparison with the large reference set that was not present in either parent, the child's CNV was considered to have arisen de novo. The software packages used to detect CNVs each employ a different algorithm to identify genomic regions of arbitrary size that have higher or lower copy number than adjacent regions. To compare CNV calls made by different packages and to identify the calls that were most likely to be biologically meaningful, we calculated t statistics and the corresponding P values for mean sample versus reference log2 copy-number ratios within each candidate deletion or duplication in comparison with the ratios outside the CNV on the same chromosome. The number of SNPs considered to be part of a CNV varies with the software and the parameters used to identify the aberrations, so we tried a range of window sizes (expressed as the number of contiguous SNPs) around the detected aberration and computed a t score for every window of the same size on the chromosome. We tested all reasonable window sizes for each candidate aberration and defined the optimal window size as the one that produced the best t score or equivalent P value for that CNV. Choice of window size for graphical display of t scores is arbitrary. We chose the optimal window size with small CNVs to maximize the t score and to make the aberration more apparent in the plot. Longer aberrations are generally associated with t scores that differ greatly from the rest of the chromosome, and, in these cases, we chose window sizes smaller than the optimal to produce smoother plots. SNP genotype calls were generated from signal intensity data with GDAS version 3.0 by use of a confidence score threshold of 0.05 for genotype accuracy. Genotypes were assessed for each putative deletion identified by CNAG or dChip. If the number of heterozygous SNPs exceeded 10% of the total within a putatively deleted segment, the deletion call was considered to be a false-positive result. Deletions were accepted as hemizygous if at least 90% of the genotype calls within the segment were either "AA" or "BB." Genotype calls within a hemizygous deletion in a child often exhibited Mendelian errors (e.g., an "AA" genotype result in the child and a "BB" genotype result in one parent), and such errors were used to determine the parental origin of the child's remaining inherited allele. Copy numbers for SNPs on the X chromosome were analyzed with dChip software33Zhao X Li C Paez JG Chin K Janne PA Chen TH Girard L Minna J Christiani D Leo C Gray JW Sellers WR Meyerson M An integrated view of copy number and allelic alterations in the cancer genome using single nucleotide polymorphism arrays.Cancer Res. 2004; 64: 3060-3071Crossref PubMed Scopus (441) Google Scholar as follows. Each male (child or father) was compared with a reference set comprising all 108 unaffected fathers included in the study. Each female (child or mother) was compared with a reference set comprising all 108 unaffected mothers included in the study. Regions of copy-number gain or loss were detected using the hidden Markov model output of dChip. A search for UPD was performed using SNP genotypes generated with GDAS version 3.0, with a confidence score threshold of 0.05. SNP genotypes for each child were compared with those of both parents, and Mendelian errors (SNPs homozygous for one allele in the child and the opposite allele in one parent) were identified. Mendelian errors are very infrequent in the absence of deletions or UPD. When such Mendelian errors were found, other SNPs within the same chromosomal region or chromosome were evaluated for the presence of isodisomy or heterodisomy. Uniparental isodisomy was identified as a stretch of homozygous SNPs in the child with exclusively maternal or exclusively paternal origin. Diagnosis of uniparental isodisomy also required confirmation of a normal disomic copy number. Uniparental heterodisomy was detected when a continuous region within a window of 200–1,000 SNPs in the child showed genotypes identical to the same chromosomal region in the mother or father. Putative CNVs identified by WGSA were validated by FISH with cytogenetic pellets prepared according to standard clinical procedures. FISH was performed with BAC or fosmid probes selected using the University of California at Santa Cruz (UCSC) Genome Browser34Kent WJ Sugnet CW Furey TS Roskin KM Pringle TH Zahler AM Haussler D The human genome browser at UCSC.Genome Res. 2002; 12: 996-1006Crossref PubMed Scopus (5930) Google Scholar and the May 2004 assembly of the human genome sequence. The genomic content of BAC inserts used for FISH confirmation of putative CNVs was verified by end sequencing, as necessary. BAC DNA was prepared as described elsewhere,35Schein J Kucaba T Sekhon M Smailus D Waterston R Marra M High-throughput BAC fingerprinting.in: Zhao S Stodolsky M Methods in molecular biology: bacterial artificial chromosomes. Humana Press, Totowa, NJ2004: 143-156Crossref Scopus (24) Google Scholar was precipitated, and was resuspended in 35 μl of Ultrapur