Microarray-Based Comparative Genomic Hybridization Using Sex-Matched Reference DNA Provides Greater Sensitivity for Detection of Sex Chromosome Imbalances than Array-Comparative Genomic Hybridization with Sex-Mismatched Reference DNA
2009; Elsevier BV; Volume: 11; Issue: 3 Linguagem: Inglês
10.2353/jmoldx.2009.080064
ISSN1943-7811
AutoresSvetlana A. Yatsenko, Chad A. Shaw, Zhishuo Ou, Amber N. Pursley, Ankita Patel, Weimin Bi, Sau Wai Cheung, James R. Lupski, A. Craig Chinault, Arthur L. Beaudet,
Tópico(s)Chromosomal and Genetic Variations
ResumoIn array-comparative genomic hybridization (array-CGH) experiments, the measurement of DNA copy number of sex chromosomal regions depends on the sex of the patient and the reference DNAs used. We evaluated the ability of bacterial artificial chromosomes/P1-derived artificial and oligonucleotide array-CGH analyses to detect constitutional sex chromosome imbalances using sex-mismatched reference DNAs. Twenty-two samples with imbalances involving either the X or Y chromosome, including deletions, duplications, triplications, derivative or isodicentric chromosomes, and aneuploidy, were analyzed. Although concordant results were obtained for approximately one-half of the samples when using sex-mismatched and sex-matched reference DNAs, array-CGH analyses with sex-mismatched reference DNAs did not detect genomic imbalances that were detected using sex-matched reference DNAs in 6 of 22 patients. Small duplications and deletions of the X chromosome were most difficult to detect in female and male patients, respectively, when sex-mismatched reference DNAs were used. Sex-matched reference DNAs in array-CGH analyses provides optimal sensitivity and enables an automated statistical evaluation for the detection of sex chromosome imbalances when compared with an experimental design using sex-mismatched reference DNAs. Using sex-mismatched reference DNAs in array-CGH analyses may generate false-negative, false-positive, and ambiguous results for sex chromosome-specific probes, thus masking potential pathogenic genomic imbalances. Therefore, to optimize both detection of clinically relevant sex chromosome imbalances and ensure proper experimental performance, we suggest that alternative internal controls be developed and used instead of using sex-mismatched reference DNAs. In array-comparative genomic hybridization (array-CGH) experiments, the measurement of DNA copy number of sex chromosomal regions depends on the sex of the patient and the reference DNAs used. We evaluated the ability of bacterial artificial chromosomes/P1-derived artificial and oligonucleotide array-CGH analyses to detect constitutional sex chromosome imbalances using sex-mismatched reference DNAs. Twenty-two samples with imbalances involving either the X or Y chromosome, including deletions, duplications, triplications, derivative or isodicentric chromosomes, and aneuploidy, were analyzed. Although concordant results were obtained for approximately one-half of the samples when using sex-mismatched and sex-matched reference DNAs, array-CGH analyses with sex-mismatched reference DNAs did not detect genomic imbalances that were detected using sex-matched reference DNAs in 6 of 22 patients. Small duplications and deletions of the X chromosome were most difficult to detect in female and male patients, respectively, when sex-mismatched reference DNAs were used. Sex-matched reference DNAs in array-CGH analyses provides optimal sensitivity and enables an automated statistical evaluation for the detection of sex chromosome imbalances when compared with an experimental design using sex-mismatched reference DNAs. Using sex-mismatched reference DNAs in array-CGH analyses may generate false-negative, false-positive, and ambiguous results for sex chromosome-specific probes, thus masking potential pathogenic genomic imbalances. Therefore, to optimize both detection of clinically relevant sex chromosome imbalances and ensure proper experimental performance, we suggest that alternative internal controls be developed and used instead of using sex-mismatched reference DNAs. Array comparative genomic hybridization (array-CGH) is a high resolution genome analysis technique used to detect DNA copy number alterations, ie, segmental genomic gains and losses. The implementation of array-CGH in clinical diagnosis and research is a fundamental step toward the elucidation of the etiology of congenital malformations frequently associated with genomic disorders,1Lupski JR Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits.Trends Genet. 1998; 14: 417-422Abstract Full Text Full Text PDF PubMed Scopus (681) Google Scholar mental retardation,2Stankiewicz P Beaudet AL Use of array-CGH in the evaluation of dysmorphology, malformations, developmental delay, and idiopathic mental retardation.Curr Opin Genet Dev. 2007; 17: 182-192Crossref PubMed Scopus (265) Google Scholar autism and behavioral abnormalities,3Beaudet AL Autism: highly heritable but not inherited.Nat Med. 2007; 13: 534-536Crossref PubMed Scopus (72) Google Scholar cancer,4Snijders AM Nowak N Segraves R Blackwood S Brown N Conroy J Hamilton G Hindle AK Huey B Kimura K Law S Myambo K Palmer J Ylstra B Yue JP Gray JW Jain AN Pinkel D Albertson DG Assembly of microarrays for genome-wide measurement of DNA copy number.Nat Genet. 2001; 29: 263-264Crossref PubMed Scopus (761) Google Scholar and benign human genome copy number variants.5Lee C Iafrate AJ Brothman AR Copy number variations and clinical cytogenetic diagnosis of constitutional disorders.Nat Genet. 2007; 39: S48-S54Crossref PubMed Scopus (315) Google Scholar,6Conrad DF Andrews TD Carter NP Hurles ME Pritchard JK A high-resolution survey of deletion polymorphism in the human genome.Nat Genet. 2006; 38: 75-81Crossref PubMed Scopus (525) Google Scholar Array-CGH compares genomic DNAs isolated from test and reference samples that are differentially labeled with red (Cy5) and green (Cy3) fluorescent dyes and competitively hybridized to known mapped segments of human genomic DNA (eg, bacterial artificial chromosomes/P1-derived artificial [BAC/PAC] or oligonucleotide probes) attached to a slide. The fluorescent signal intensity of the two fluorochromes at each spot on the microarray is proportional to the amount of test and reference DNA samples bound to the DNA sequence at a given genomic position. The presence of chromosomal imbalance can be detected and quantified by calculating the ratio of signal intensities of test DNA versus reference DNA. For statistical purposes, these ratios are usually converted to a log2(Cy5/Cy3) ratio for interpretation and analysis (see Supplemental Table 1 at http://jmd.amjpathol.org). The log2(Cy5/Cy3) scaling has the consequence of centering the ratios at approximately zero, making DNA copy number losses appear negative, and copy number gains positive. In general, a patient's test genome may contain 0, 1, 2, 3, or more copies of a genomic interval, when compared with the reference diploid genome. The ability of array-CGH to detect single copy changes (ie, ratios 1:2, 3:2, 0:1, 2:1) has been previously demonstrated.7Bignell 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 (303) Google Scholar,8Pinkel D Albertson DG Comparative genomic hybridization.Annu Rev Genomics Hum Genet. 2005; 6: 331-354Crossref PubMed Scopus (179) Google Scholar In contrast, distinguishing homozygous deletions, triplications, and other amplifications (ie, ratios 0:2, 4:2, 3:1, 5:2, and so forth), as well as ascertainment of DNA copy numbers in samples with a mixed population of abnormal and normal cells, such as in tumor tissue or a blood specimen with mosaicism, remains a challenge. The use of array-CGH is expanding rapidly as a tool for the identification of genomic copy number abnormalities in patients.9Bejjani BA Saleki R Ballif BC Rorem EA Sundin K Theisen A Kashork CD Shaffer LG Use of targeted array-based CGH for the clinical diagnosis of chromosomal imbalance: is less more?.Am J Med Genet A. 2005; 134: 259-267Crossref PubMed Scopus (181) Google Scholar10Cheung SW Shaw CA Yu W Li J Ou Z Patel A Yatsenko SA Cooper ML Furman P Stankiewicz P Lupski JR Chinault AC Beaudet AL Development and validation of a CGH microarray for clinical cytogenetic diagnosis.Genet Med. 2005; 7: 422-432Crossref PubMed Scopus (225) Google Scholar11Choe J Kang JK Bae CJ Lee DS Hwang D Kim KC Park WY Lee JH Seo JS Identification of origin of unknown derivative chromosomes by array-based comparative genomic hybridization using pre- and postnatal clinical samples.J Hum Genet. 2007; 52: 934-942Crossref PubMed Scopus (15) Google Scholar12Emanuel BS Saitta SC From microscopes to microarrays: dissecting recurrent chromosomal rearrangements.Nat Rev Genet. 2007; 8: 869-883Crossref PubMed Scopus (83) Google Scholar13Shaffer LG Bejjani BA Medical applications of array CGH and the transformation of clinical cytogenetics.Cytogenet Genome Res. 2006; 115: 303-309Crossref PubMed Scopus (80) Google Scholar14Lu X Shaw CA Patel A Li J Cooper ML Wells WR Sullivan CM Sahoo T Yatsenko SA Bacino CA Stankiewicz P Ou Z Chinault AC Beaudet AL Lupski JR Cheung SW Ward PA Clinical implementation of chromosomal microarray analysis: summary of 2513 postnatal cases.PLoS ONE. 2007; 2: e327Crossref PubMed Scopus (182) Google Scholar15Ou Z Kang S-HL Shaw CA Carmack CE White LD Patel A Beaudet AL Cheung SW Chinault AC Bacterial artificial chromosome-emulation oligonucleotide arrays for targeted clinical array-comparative genomic hybridization analyses.Genet Med. 2008; 10: 278-289Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar Abnormality rates of 10 to 15% are widely reported for heterogeneous patient populations typically studied by cytogenetic methods in the past.2Stankiewicz P Beaudet AL Use of array-CGH in the evaluation of dysmorphology, malformations, developmental delay, and idiopathic mental retardation.Curr Opin Genet Dev. 2007; 17: 182-192Crossref PubMed Scopus (265) Google Scholar,16Cheung SW Shaw C Scott DA Patel A Sahoo T Bacino CA Pursley A Li J Erickson R Gropman AL Miller DT Seashore MR Summers AM Stankiewicz P Chinault AC Lupski JR Beaudet AL Sutton VR Microarray-based CGH detects chromosomal mosaicism not revealed by conventional cytogenetics.Am J Med Genet A. 2007; 143A: 1679-1686Crossref PubMed Scopus (146) Google Scholar,17Shaffer LG Bui TH Molecular cytogenetic and rapid aneuploidy detection methods in prenatal diagnosis.Am J Med Genet C Semin Med Genet. 2007; 145C: 87-98Crossref PubMed Scopus (107) Google Scholar Sex chromosome aneuploidy, microdeletion, and microduplication abnormalities are relatively common, and are of special interest because of their frequent association with congenital anomalies, mental retardation syndromes, infertility, and lethal conditions. Abnormalities involving the X and Y chromosomes can be quite complex, are frequently present in mosaic form, and are associated with a variable phenotype between males and females, the latter complicated by random X-inactivation. Identification of sex chromosome rearrangements is particularly important for adequate genetic counseling. In array-CGH experiments the measurement of DNA copy number of sex chromosomal regions depends heavily on the sex of the patient and reference DNA used. Although the difference in copy number between X and Y chromosomes in array-CGH studies using sex-mismatched reference DNAs serves as a positive experimental control,18Shaffer LG Beaudet AL Brothman AR Hirsch B Levy B Martin CL Mascarello JT Rao KW Working Group of the Laboratory Quality Assurance Committee of the American College of Medical Genetics: microarray analysis for constitutional cytogenetic abnormalities.Genet Med. 2007; 9: 654-662Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar the sensitivity of various CGH platforms to detect constitutional sex chromosome imbalances when using sex-mismatched as compared with sex-matched references has not been systematically evaluated. This study evaluates the sensitivity of two array-CGH platforms (BAC/PAC and oligonucleotide microarrays) to detect sex chromosome abnormalities using sex-matched versus sex-mismatched reference DNA samples and demonstrates the importance of experimental design for the interpretation of array-CGH results in clinical diagnosis. The samples analyzed were selected from 6400 patients whose blood samples were submitted to the Baylor College of Medicine Clinical Cytogenetics Laboratory for array-CGH analysis between August 2005 and March 2007. Patient genomic DNAs were isolated from peripheral blood using a PureGen kit (Gentra Systems, Minneapolis, MN) according to the manufacturer's protocol. Genomic DNA obtained from whole blood of either a normal male or female individual, was used in all experiments as reference DNA. Initial array-CGH analyses with sex-matched reference DNA were performed using targeted BAC/PAC-based microarray-CGH (Baylor College of Medicine, Version 5 or Version 6.1). The latter microarray contained a total of ∼1400 probes spotted in duplicate, and included 120 and 15 probes specific for the X and Y chromosomes, respectively, and four probes from the X/Y short arm pseudoautosomal region (PAR1) and one from the long arm pseudoautosomal region (PAR2). Among the X- and Y-specific probes, approximately half were positioned within clinically relevant and subtelomeric targeted regions, whereas the other half represented backbone clones at a density of one probe per 5 Mb. Labeled genomic DNA from the patient and sex-matched reference DNA were hybridized to a BAC/PAC microarray as previously described.10Cheung SW Shaw CA Yu W Li J Ou Z Patel A Yatsenko SA Cooper ML Furman P Stankiewicz P Lupski JR Chinault AC Beaudet AL Development and validation of a CGH microarray for clinical cytogenetic diagnosis.Genet Med. 2005; 7: 422-432Crossref PubMed Scopus (225) Google Scholar,16Cheung SW Shaw C Scott DA Patel A Sahoo T Bacino CA Pursley A Li J Erickson R Gropman AL Miller DT Seashore MR Summers AM Stankiewicz P Chinault AC Lupski JR Beaudet AL Sutton VR Microarray-based CGH detects chromosomal mosaicism not revealed by conventional cytogenetics.Am J Med Genet A. 2007; 143A: 1679-1686Crossref PubMed Scopus (146) Google Scholar Array-CGH analyses with sex-mismatched reference DNA using the BAC/PAC microarray platform were performed for 22 patients selected for this study. In addition, 8 patients of the 22 had microarray analysis using a commercially available whole genome oligonucleotide-based array-CGH (Agilent Human Genome CGH Microarray Kit 244A; Agilent Technologies, Inc., Santa Clara, CA). According to the manufacturer, the 244A format has extensive probe coverage including coding and noncoding regions, well-known genes, promoters, miRNAs, and telomeric regions. It contains ∼11,000 and 1300 oligonucleotide 60-mer probes specific for X and Y chromosomes, respectively. Probe design has been optimized for maximum sensitivity and specificity within unique sequence.7Bignell 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 (303) Google Scholar The 244A platform has no coverage of the X/Y pseudoautosomal regions. Whole genome array-CGH with the 244A array was performed according to the manufacturer's protocol. In all array-CGH platforms, the accurate identification of copy number gains and losses requires a combination of visual and computational analysis. For the BAC/PAC microarrays, the fluorescent signal intensities were scanned using a GenePix 4000B microarray scanner (Axon Instruments, Union City, CA) and quantified using BlueFuse for Microarrays version 3.2 software (BlueGnome Ltd, Cambridge, UK). Although the same hybridization conditions apply to all probes on the array-CGH, the hybridization signal intensity is affected by a number of factors. In practice, the performance of the DNA labeling reaction is a particularly critical variable. Even if there is no copy number change, signal intensity and log2(Cy5/Cy3) ratios for each individual probe can be variable among a consecutive series of experiments. To correct for experimental artifacts, array-CGH data are normalized, so that the ratio for a diploid genome is set to a standard value, usually 1.0 on a linear scale (0.0 on a log2 scale), and threshold values are ascertained for loss, no change, and gain status relative to the diploid reference DNA (see Supplemental Table 1 at http://jmd.amjpathol.org). Threshold ratios were established experimentally for a particular array-CGH platform by validating DNA samples from individuals with known chromosomal gains and losses.19Ng G Huang J Roberts I Coleman N Defining ploidy-specific thresholds in array comparative genomic hybridization to improve the sensitivity of detection of single copy alterations in cell lines.J Mol Diagn. 2006; 8: 449-458Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar The typical mean normal ratio for BAC/PAC-derived array-CGH has been established at 0.0 ± 0.2 on the log2 scale, indicating equal copy number of test and reference sample DNA. A change in log2(Cy5/Cy3) ratio greater than +0.2 or less than −0.2 threshold range typically suggests the possibility of a true gain or loss in DNA dosage at the given interrogating probe locus. Additional computation can refine the cutoff values and generate P values for individual patient results.19Ng G Huang J Roberts I Coleman N Defining ploidy-specific thresholds in array comparative genomic hybridization to improve the sensitivity of detection of single copy alterations in cell lines.J Mol Diagn. 2006; 8: 449-458Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar The mean log2 ratio (patient/reference) of intensity for each single probe was integrated for data processing. Raw data from all probes and dye reversal pairs were combined for subsequent comparative statistical analysis.20Shaw CJ Shaw CA Yu W Stankiewicz P White LD Beaudet AL Lupski JR Comparative genomic hybridisation using a proximal 17p BAC/PAC array detects rearrangements responsible for four genomic disorders.J Med Genet. 2004; 41: 113-119Crossref PubMed Scopus (63) Google Scholar For each sex-matched experiment, a chromosomal region was classified as containing a gain (likely duplicated) or loss (likely deleted) when the value for the log2(Cy5/Cy3) ratio of an interrogating BAC/PAC probe or oligonucleotide probe fell outside the fixed threshold (+0.2 and −0.2). The T-statistics (>3.0) and P values ( log2(Cy5/Cy3) > +0.2; test/reference ratio is 2:1, see Supplemental Table 1 at http://jmd.amjpathol.org] and all Y-specific probes showed a loss [log2(Cy5/Cy3) < −0.2; test/reference ratio is 0:1, see Supplemental Table 1 at http://jmd.amjpathol.org]. In contrast, a male array-CGH profile is considered normal when log2(Cy5/Cy3) ratios for all X-specific probes show a loss [−1.0 < log2(Cy5/Cy3) < −0.2; test/reference ratio is 1:2, see Supplemental Table 1 at http://jmd.amjpathol.org] and all Y-specific probes show a gain [log2(Cy5/Cy3) > +0.2; test/reference ratio is 1:0, see Supplemental Table 1 at http://jmd.amjpathol.org]. Thus, in sex-mismatched CGH, variable test/reference ratios occur depending on the genomic region interrogated; 2:2 for autosomal probes, 2:1 or 1:2 for X-specific, and 1:0 or 0:1 for Y-specific probes. For sex-mismatched experiments a loss (deletion) or a gain (duplication) of the chromosome region was suspected if the probe log2(Cy5/Cy3) ratios were lower or higher than the expected values for the X and Y copy differences. In sex-mismatched experiments, the T-statistics with a null hypothesis of expected mean zero values and the corresponding P values cannot be used consistently as criteria for sex chromosome-specific probes because of the statistically significant difference for all sex chromosome-specific probes regardless of the software used. An interpretation is rendered based on graphical profiles of the hybridizations and manual analysis of signal intensity data. Oligonucleotide microarray data were obtained using Agilent Feature Extraction software 9.5.3 and imported into Agilent CGH analytics 3.4.4 software for analysis. Detection of losses and gains were based on the log2 ratio (less than −0.3 or greater than +0.3) and z-score algorithm (cutoff 2.5) with a moving average of 50 kb (Agilent Technologies). Each abnormality tentatively identified by array-CGH analysis (BAC/PAC and oligonucleotide) was independently assessed experimentally using locus-specific FISH and/or conventional G-banding chromosome analyses. DNAs from BAC and PAC clones spotted on the array were isolated using the Perfectprep mini kit (Eppendorf, Hamburg, Germany) and directly labeled using Spectrum Red or Spectrum Green dUTPs (Abbott/Vysis, Inc., Des Plaines, IL). FISH analyses were performed according to a standard protocol on metaphase chromosomes or interphase nuclei from lymphocyte cultures of peripheral blood. At least 10 metaphase cells and 50 interphase cells were scored to detect deletions or duplications, respectively. A minimum of 100 cells were scored in all cases with suspected mosaicism. Confirmatory FISH analysis for each male patient was accompanied by control hybridization on a normal female sample. Chromosome analysis was performed using standard cytogenetic methods. Array-CGH analysis using sex-matched reference DNA identified 122 patients from 117 families (51 phenotypic females and 71 phenotypic males) with constitutional imbalances involving the sex chromosomes (Table 1, Table 2). These 122 cases accounted for ∼20% of all abnormal cases identified in our clinical laboratory among the screened cohort during the interval of study. Patients with a single BAC clone gain or loss of unclear clinical significance were not included in this analysis. Routine GTG-banding studies performed at different laboratories before array-CGH revealed a normal karyotype in 48 of the 122 patients. Twelve patients had an abnormal karyotype; however, the genomic content or extent of imbalance of X or Y was not determined. Chromosome analysis had not been performed in 62 patients before array-CGH analysis but was completed concurrently for 37 of these cases. Of the 97 patients with prior or concurrent karyotype, 82 were reported as normal. Mosaicism was detected in 19 of the 122 patients (16%).Table 1Summary of Cytogenetic and Array-CGH Results for Female Representative PatientsCategory of sex chromosome abnormalitiesTotal no. of patientsRepresentative patient (Pt)Array-CGH plotsMosaicismInterpretation of sex-matched study/karyotypeInterpretation of sex-mismatched experimentsConcordanceNumber of interrogating BAC/PAC probesPoly-X syndrome one cell line4Pt 1Figure 1, C and DNo47,XXX47,XXX suspected?120Poly-X syndrome multiple cell lines2Pt 2∼40%47,XXX[13]/46,XX[17]46,XX−120Monosomy X one cell line2Pt 3No45,X45,X+120Monosomy X multiple cell lines4Pt 4Figure 1, E and F∼50%45,X[24]/46,XX[26]46,XX−120Apparent X deletions8Pt 5No46,X,del(X) (p11.4)46,X,del(X) (p11.4)+16Apparent X duplications4Pt 6No46,X,dup(X) (q26q27)46,X,dup(X) (q26q27)+9Small X deletions7Pt 7Figure 2, A and BNo46,X,del(X) (q21q21)46,X,del(X) (q21q21)+2Small X duplications11Pt 8No46,X,dup(X) (p22p22)46,X,dup(X) (p22p22) suspected?3Pt 9Figure 2, C and DNo46,X,dup(X) (q28q28)46,X,dup(X) (q28q28) suspected?4Complex X abnormalities10Pt 10Figure 2, E and F∼55%45,X[55]/46,X,dic(X) (q13)[45]46,X,del(X) (q13)−120Complex Y abnormalities1Pt 11No46,X,i(Y)(q10)del(Y)(p10) or i(Y)(q10) suspected?8 Open table in a new tab Table 2Summary of Cytogenetic and Array-CGH Results for Male Representative PatientsCategory of sex chromosome abnormalitiesTotal no. of patientsRepresentative patient (Pt)Array-CGH plotsMosaicismInterpretation of sex-matched study/karyotypeInterpretation of sex-mismatched experimentsConcordanceNumber of interrogating BAC/PAC probesPoly-X syndrome one cell line7Pt 12No47,XXY47,XXY+120Poly-X syndrome multiple cell lines2Pt 13∼42%47,XXY[21]/46,XY[29]46,XY−120Monosomy X multiple cell lines2Pt 14∼33%45,X[10]/46,XY[20]46,XY−120Disomy Y7Pt 15Figure 3, C and DNo47,XYY46,XYY suspected?15Apparent X deletions3Pt 16No46,Y,del(X) (p22p22)46,Y,del(X) (p22p22) suspected+12Apparent X duplications7Pt 17No46,Y,der(7)t(X;7) (p22.31;q35)46,Y,der(7)t(X;7) (p22.31;q35)+16Complex X abnormalities2Pt 18No46,Y,der(X)del(X) (p22) dup(X)(q27)46,Y,der(X)del(X)(p22) dup(X)(q27)+20Small X deletions9Pt 19Figure 3, E and FNo46,Y,del(X) (p22p22)46,XY−3Small X duplications23Pt 20Figure 3, G and HNo46,Y,dup(X) (q28q28)46,Y,dup(X)(q28q28)+4Complex Y abnormalities5Pt 21No45,X,i(Y)(p10)45,X,del(Y)(q10)?8Complex X and Y abnormalities2Pt 22No46,X,der(X)t(X;Y)46,X,der(X)t(X;Y)?6 Open table in a new tab The majority of patients were referred for clinical indications including nonsyndromic developmental delay and mental retardation, behavioral abnormalities, dysmorphic features, and multiple congenital anomalies. A subgroup (∼15%) of patients had diagnoses associated with X or Y chromosome rearrangements or an established carrier status. Approximately 36% of patients were positive for submicroscopic deletions or duplications of either the X or Y chromosome, and in some of the patients, cryptic imbalances were present as a part of a complex rearrangement. Duplication of a genomic region encompassing the MECP2 gene (Lubs X-linked mental retardation syndrome, MRXSL; OMIM no.300260) was found as the most frequent familial aberration affecting males (n = 19). Approximately 25% of patients were identified to have common deletions and duplications associated with well-known sex chromosome genomic disorders such as X-linked ichthyosis (STS, OMIM no. 308100), Duchenne muscular dystrophy (DMD, OMIM no. 310200), Kallmann syndrome (KAL1, MIM no. 308700), Pelizaeus-Merzbacher disease (PMD, OMIM no. 312080), or Rett syndrome (RTT, OMIM no. 312750); however the size of the segmental aneusomy was variable between the patients. Rare deletions and duplication events of the X and Y chromosome were detected in ∼20% of patients. The observed sex chromosome abnormalities were classified into several categories based on the number of probes involved: poly X syndrome (gain with all X-specific probes), monosomy X (one copy with all X probes), disomy Y (two copies with all Y-specific probes), apparent segmental aneusomies because of duplications/deletions (more than three consecutive BAC clones showing a gain or loss) and complex rearrangements (co-existence of loss and gain in copy number with probes representing different segments of the X or Y chromosome). Patients with segmental aneusomies were subdivided into two major categories depending on the number of consecutive interrogating probes that reveal either gains or losses. Patients with imbalances detected by four or more consecutive probes were included in the category apparent segmental aneusomies, whereas the category small duplications/deletions comprises patients in whom abnormalities were identified by less than four consecutive BAC probes. Deletions or duplications identified by four or more consecutive probes produce a pattern that can be more easily visualized on a hybridization profile, whereas imbalances detected by less than four probes are not always obvious on an array CGH graphical profile. Because interpretation of array CGH analyses using sex-mismatched reference DNA was based primarily on visual examination of hybridization profiles, we sought to determine the sensitivity of array CGH as a function of the number of consecutive data points observed from the interrogated probes. To evaluate the ability to detect sex chromosome abnormalities by array-CGH using sex-mismatched reference DNA, we selected 22 patients with representative sex chromosome imbalances. The results of sex-mismatched versus sex-matched experiments are shown in Tables 1and 2 for female and male patients, respectively. An array-CGH experimental design using either sex-mismatched or sex-matched reference DNA results in two normal array-CGH profiles for a test female versus reference male DNA (Figure 1A) and a test female versus reference female DNA (Figure 1B). The array-CGH analysis of 11 samples from female patients (Table 1) were concordant in four cases (patients 3, 5, 6, and 7) performed with either sex-mismatched or sex-matched reference DNA. For the remaining seven patients, BAC/PAC array-CGH results were eithe
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