Detection Of Chromosomal Imbalances Research Articles

Microarray‐based comparative genomic hybridization analysis in neonates with congenital anomalies: detection of chromosomal imbalances

ObjectiveTo identify chromosomal imbalances by whole‐genome microarray‐based comparative genomic hybridization (array‐CGH) in DNA samples of neonates with congenital anomalies of unknown cause from a birth defects monitoring program at a public maternity hospital. MethodsA blind genomic analysis was performed retrospectively in 35 stored DNA samples of neonates born between July of 2011 and December of 2012. All potential DNA copy number variations detected (CNVs) were matched with those reported in public genomic databases, and their clinical significance was evaluated. ResultsOut of a total of 35 samples tested, 13 genomic imbalances were detected in 12/35 cases (34.3%). In 4/35 cases (11.4%), chromosomal imbalances could be defined as pathogenic; in 5/35 (14.3%) cases, DNA CNVs of uncertain clinical significance were identified; and in 4/35 cases (11.4%), normal variants were detected. Among the four cases with results considered causally related to the clinical findings, two of the four (50%) showed causative alterations already associated with well‐defined microdeletion syndromes. In two of the four samples (50%), the chromosomal imbalances found, although predicted as pathogenic, had not been previously associated with recognized clinical entities. ConclusionsArray‐CGH analysis allowed for a higher rate of detection of chromosomal anomalies, and this determination is especially valuable in neonates with congenital anomalies of unknown etiology, or in cases in which karyotype results cannot be obtained. Moreover, although the interpretation of the results must be refined, this method is a robust and precise tool that can be used in the first‐line investigation of congenital anomalies, and should be considered for prospective/retrospective analyses of DNA samples by birth defect monitoring programs.

Microarray-based comparative genomic hybridization analysis in neonates with congenital anomalies: detection of chromosomal imbalances

ObjectiveTo identify chromosomal imbalances by whole-genome microarray-based comparative genomic hybridization (array-CGH) in DNA samples of neonates with congenital anomalies of unknown cause from a birth defects monitoring program at a public maternity hospital. MethodsA blind genomic analysis was performed retrospectively in 35 stored DNA samples of neonates born between July of 2011 and December of 2012. All potential DNA copy number variations detected (CNVs) were matched with those reported in public genomic databases, and their clinical significance was evaluated. ResultsOut of a total of 35 samples tested, 13 genomic imbalances were detected in 12/35 cases (34.3%). In 4/35 cases (11.4%), chromosomal imbalances could be defined as pathogenic; in 5/35 (14.3%) cases, DNA CNVs of uncertain clinical significance were identified; and in 4/35 cases (11.4%), normal variants were detected. Among the four cases with results considered causally related to the clinical findings, two of the four (50%) showed causative alterations already associated with well-defined microdeletion syndromes. In two of the four samples (50%), the chromosomal imbalances found, although predicted as pathogenic, had not been previously associated with recognized clinical entities. ConclusionsArray-CGH analysis allowed for a higher rate of detection of chromosomal anomalies, and this determination is especially valuable in neonates with congenital anomalies of unknown etiology, or in cases in which karyotype results cannot be obtained. Moreover, although the interpretation of the results must be refined, this method is a robust and precise tool that can be used in the first-line investigation of congenital anomalies, and should be considered for prospective/retrospective analyses of DNA samples by birth defect monitoring programs.

Application of array comparative genomic hybridization (array- CGH) for detection of chromosomal imbalances in children with developmental delay/congenital malformations in Saudi Arabia

Background Microarray–based Comparative Genomic Hybridization (a-CGH) has enabled wide investigation of the genome at high resolution and has been implemented in different centers as a clinical diagnostic tool. Chromosomal imbalances are implicated in the etiology of Developmental Delay (DD)/Intellectual Disability (ID)/Congenital Malformations. However, most of these cases may not be diagnosed by conventional cytogenetic techniques. We aimed to establish a-CGH technique and assess its potential as a diagnostic tool for chromosomal imbalances and to detect known and novel chromosomal aberrations in patients with DD/ congenital malformations.

Detection of chromosomal imbalances using combined MLPA kits in patients with syndromic intellectual disability

Intellectual disability (ID) is a common disorder, with major consequences for individual, family and society. Due to clinical and genetic heterogeneity of ID, in about 50% of cases an etiologic diagnosis cannot be established. The aim of this study was to evaluate the ability of a combination of MLPA kits to establish the diagnosis in 369 patients with syndromic ID and normal or uncertain routine karyotype results. All patients were assessed for chromosome imbalance using SALSA MLPA P064 or P096 kits, if the phenotype was suggestive of a microdeletion syndrome (subgroup A 186 patients), or subtelomeric P036 and P070 kits, if the phenotype was not suggestive of a microdeletion syndrome or if the result of the standard karyotype was uncertain (subgroup B 183 patients). Abnormal results detected by these kits were further characterized using appropriate follow-up MLPA kits (Telomere Follow-up set, P029-A1, P250-B2, ME028-B1). In subgroup A we identified 25 patients with microdeletions (13.4%). Using subtelomere screening and follow-up kits in subgroup B we detected cryptic rearrangements in 7.5% cases and identified the origin of the unknown material noticed in the standard karyotype in 10 out of 11 patients. Summarizing data from the two groups, the combined use of MLPA kits led to the diagnosis in 10.6% (38/358) patients with normal karyotype. Using follow-up MLPA kits allowed us both to confirm abnormalities and to determine their size, which facilitated the interpretation of the clinical significance of these rearrangements. For laboratories that do not have yet access to microarray technology, using several MLPA kits represents an effective strategy for establishing the diagnosis in ID patients.

Detection of chromosomal imbalance in ependymoma by comparative genomic hybridization

To investigate genomic DNA imbalances in ependymomas (EDMs) and their correlations with the tumor histological types, grades, locations, patients' gender and age. Chromosomal gains and losses in 16 cases of EDM were analyzed using comparative genomic hybridization. Chromosomal regional gain and loss were found in 15 and 13 of 16 EDM cases respectively including totally 24 regional gains and 19 regional losses in all the tumors studied. Both regional gains and losses were mostly seen in myxopapillary EDMs (MPE, WHO grade I), more commonly seen in cellular EDMs (CE, WHO grade II) and tanycytic EDMs (TE, WHO grade II) than in anaplastic EDMs (AE, WHO grade III). Some of the regional gains and losses appeared only in one subtype of MPE, CE, TE and AE cases resulting in development of specific imbalance profiles of certain subtype in these cases. MPE, CE and TE often had +7. Chromosomal +5 occurred only in MPE and CE, and -22q was only seen in CE and TE. AE frequently had +1q, but none had +5, +7, -4q, -19q and -22q. The frequencies of any regional gain or loss were not affected by patients' genders (P > 0.05). Chromosomal +1q and +7p happened predominantly in intracranial EDMs with an averagely onset age of <or= 30 years, and +7 was only detected in spinal EDMs of patients over 30 years old, the differences were statistically significant (P < 0.05). The frequencies of chromosomal imbalances in EDMs decrease as the tumor grade increases. Characteristic chromosomal imbalances in each subtype may play an important role in determination of histological phenotypes and tumor grades. Chromosomal +1q, +5, +7p, +7, -4q, -19q and -22q are alterations which may dictate the biological behaviors of these tumors and the patients' prognosis.

Recombination mapping: a universal molecular karyotyping method for preimplantation genetic diagnosis of inherited disease

OBJECTIVE: Preimplantation genetic diagnosis (PGD) is now possible for any known single gene disorder using a combination of mutation detection and closely linked markers or multiple linked markers alone. However, these methods generally require patient or disease specific test development. Moreover, chromosome aneuploidy, which is prevalent in the human preimplantation embryo, is only detectable for the chromosome hosting the mutation or a limited set of chromosomes. The objective of this study is to demonstrate proof of principle for a novel approach to create a molecular karyotype based on mapping recombination patterns for all chromosomes.DESIGN: Prospective, randomized, and blinded.MATERIALS AND METHODS: DNA from cell cultures (Coriell) derived from a family (parents and five children) with known mutations in CFTR on chromosome 7 was analysed using 650k (2kb SNP spacing) and 370k (5kb SNP spacing) microarrays and the data analyzed by BeadStudio Software v3.1 after scanning on an Illumina 500G. Call rates for pure DNA ranged from 98.9 to 99.1%, with reproducibility of >99%. SNP data were analysed using proprietary software to determine recombination maps for each chromosome (particularly chromosome 7).RESULTS: Genotype analysis and recombination mapping of both parental chromosomes 7 accurately predicted carrier/affected status in each of the 5 children. This was confirmed by a combination of direct mutation detection for delta F508 and 3 intragenic markers.CONCLUSIONS: Recombination mapping will revolutionise PGD by providing a universal method for enhanced linkage analysis to detect any single gene disorder without the need for family-specific test development. This approach simultaneously allows detection of chromosomal imbalance across all chromosomes (including uniquely the parental origin and phase of origin of any trisomy - information not available by current methods). Furthermore, genome-wide analysis automatically includes HLA genotype information (facilitating cord blood stem cell transplantation if needed) and, for the first time, allows the assessment of genetic predisposition to diseases involving multiple genetic loci, such as diabetes. OBJECTIVE: Preimplantation genetic diagnosis (PGD) is now possible for any known single gene disorder using a combination of mutation detection and closely linked markers or multiple linked markers alone. However, these methods generally require patient or disease specific test development. Moreover, chromosome aneuploidy, which is prevalent in the human preimplantation embryo, is only detectable for the chromosome hosting the mutation or a limited set of chromosomes. The objective of this study is to demonstrate proof of principle for a novel approach to create a molecular karyotype based on mapping recombination patterns for all chromosomes. DESIGN: Prospective, randomized, and blinded. MATERIALS AND METHODS: DNA from cell cultures (Coriell) derived from a family (parents and five children) with known mutations in CFTR on chromosome 7 was analysed using 650k (2kb SNP spacing) and 370k (5kb SNP spacing) microarrays and the data analyzed by BeadStudio Software v3.1 after scanning on an Illumina 500G. Call rates for pure DNA ranged from 98.9 to 99.1%, with reproducibility of >99%. SNP data were analysed using proprietary software to determine recombination maps for each chromosome (particularly chromosome 7). RESULTS: Genotype analysis and recombination mapping of both parental chromosomes 7 accurately predicted carrier/affected status in each of the 5 children. This was confirmed by a combination of direct mutation detection for delta F508 and 3 intragenic markers. CONCLUSIONS: Recombination mapping will revolutionise PGD by providing a universal method for enhanced linkage analysis to detect any single gene disorder without the need for family-specific test development. This approach simultaneously allows detection of chromosomal imbalance across all chromosomes (including uniquely the parental origin and phase of origin of any trisomy - information not available by current methods). Furthermore, genome-wide analysis automatically includes HLA genotype information (facilitating cord blood stem cell transplantation if needed) and, for the first time, allows the assessment of genetic predisposition to diseases involving multiple genetic loci, such as diabetes.

Targeted Array CGH

Targeted Array CGH

CGH-Profiler: Data mining based on genomic aberration profiles

BackgroundCGH-Profiler is a program that supports the analysis of genomic aberrations measured by Comparative Genomic Hybridisation (CGH). Comparative genomic hybridisation (CGH) is a well-established, molecular cytogenetic method that allows the detection of chromosomal imbalances in entire genomes. This technique is widely used in routine molecular diagnostics. Typically, chromosomal imbalances are described in a complex syntax based on the International Standard for Cytogenetic Nomenclature (ISCN). This semantic description of chromosomal imbalances hinders a large-scale statistical analysis across different experiments, e.g. for finding aberration patterns associated with a particular disease type or state.ResultsCGH-Profiler circumvents the semantic ISCN description by importing data from different CGH system vendors and by directly transferring the data into a table format that is readily accessible for subsequent statistical analysis. CGH-profiler comes with different consistency checks, calculates various statistics and automatically assigns a median copy number ratio to each chromosomal band.Import of CGH profiles from different CGH system vendors is already supported; its extension to other systems can be readily achieved through Perl scripts.CGH profiler can also be used to analyse comparative expressed sequence hybridisation (CESH) data. CESH reveals gene expression patterns according to chromosomal locations in a similar manner as CGH detects chromosomal imbalances.ConclusionCGH-Profiler is a useful tool for processing of CGH and CESH data.

Detection of chromosomal imbalances in retinoblastoma by matrix‐based comparative genomic hybridization

The genetic hallmark of retinoblastoma is mutation or deletion of the RB1 gene, whereas other genetic alterations that are also required are largely unknown. To screen for genomic imbalances on a genomewide level, we studied a series of 17 primary retinoblastomas by matrix-based comparative genomic hybridization (matrix-CGH). The matrix-CGH chip contained 6,000 immobilized genomic DNA fragments covering the human genome, with an average resolution of about 500 kb. The most frequent imbalances detected were gains on chromosome arms 1q (12 of 17), 6p (10 of 17), 2p (5 of 17), and 19q (4 of 17) and loss on 16q (7 of 17). Candidate regions could be narrowed to small intervals by the identified minimally overlapping regions on 1q22, 1q32.1q32.2, 2p24.1, and 6p21.33-p21.31. Furthermore, two as-yet-unknown high-level amplifications were detected, each in a single patient, on chromosome bands 1p34.2 and 1p33. Thus, this study identified new chromosomal regions and therefore potential candidate genes that may play a role in retinoblastoma.

Microarray-based CGH in cancer.

AbstractThe detection of chromosome imbalances in malignancies has been a popular and useful approach in understanding key genetic loci and the molecular pathways of carcinogenesis. Alterations in gene dosage constitutes a major mechanism that leads to an imbalance in the gene products of oncogenes or tumor suppressor genes, resulting in the loss of normal cell division. One of the more robust technologies employed for determining alterations in gene dosage has been termed “comparative genomic hybridization” (CGH). CGH is a method that measures relative DNA copy differences between hybridized test DNA labeled with one fluorophore and reference DNA labeled in a spectrally distinct fluorophore. It was developed over a decade ago by Kallioniemi et al., who applied it to the study of copy number changes, in cancer cell lines and bladder tumors (1). Two limitations of CGH technology are the inability to detect balanced changes, such as translocations and inversions, and the necessity for tumor purity (≥80%). Collectively, CGH studies done over the past decade have demonstrated that certain regions are more frequently altered. Moreover, the patterns of alterations seen in different cancers are not the same. As tumors progress, the frequency of copy number changes increase so that as much as 50% of the genome of solid tumors can be aberrant. Most of these changes are secondary effects because of the cell’s inability to replicate and segregate its chromosomes in a normal fashion.KeywordsComparative Genomic HybridizationCopy Number ChangePolymerase Chain Reaction MachineMicroarray Comparative Genomic HybridizationComparative Genomic Hybridization StudyThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Detection of chromosomal imbalances in spinal meningiomas by comparative genomic hybridization.

Little is known about genetic mutations during the malignant progression of spinal meningiomas. This study investigated genomic changes across the entire genome in spinal meningioma samples to determine possible mechanism(s) of tumorigenesis. Paraffin-embedded tissue sections of 16 spinal meningiomas were analyzed by the comparative genomic hybridization (CGH) technique. Lymphocytes of the patients were evaluated as controls. Genomic change was detected in 11 samples. Complete or partial loss of chromosome 22 was the most commonly seen abnormality in eight cases. Chromosome losses on 1p, 9p, and 10q and gains on 5p and 17q were the other abnormalities. These changes are all frequently seen in meningiomas, but are mostly specific to atypical and anaplastic meningiomas. However, in the present study, copy number changes on chromosomes 9p (3 samples), 17q (2 samples), and 1p (2 samples) were seen even in the benign tumors. Our results suggest that in addition to the neurofibromatosis type 2 tumor suppressor gene, other cancer-related genes located on 1p, 9p, 10q, and 17q might be involved in the etiology of spinal meningiomas.

Detection of chromosomal imbalances in papillary bladder tumors by comparative genomic hybridization

Objectives. To identify those genetic alterations that are associated with bladder cancer invasion and progression. Methods. A total of 30 specimens of transitional cell carcinoma of the bladder were analyzed by comparative genomic hybridization. The results were compared and summarized with previously reported studies. Results. The most frequent chromosome changes detected in our series of tumors were losses in 9q, 9p, 8p, and 11p and gains in 8q, 1q, 20q, and 11q. Three regions of deletion on chromosome 9 were delineated, at 9p21-p22, 9q13-q22, and 9q31-q34. Gains in 1q and losses on 11p were significantly more frequent in pT1G2 tumors than in superficial (pTa) ones. In our study, the most striking differences were seen between pT1G3 and pT1G2 tumors. Gains on 10p and 6p and losses at 5q, 6q, and 18q were significantly more frequent in the former. Conclusions. A summary of our results and those available from published reports suggest that several groups of chromosomal imbalances may be associated with specific steps along bladder cancer progression. These genetic changes assume two different patterns: those that are shared, but are more intensive in one stage than in the other, and those such as a gain on 3p that are unique to invasive tumors.

A database system for comparative genomic hybridization analysis.

Comparative genomic hybridization (CGH) is a molecular cytogenetic analysis method that allows the detection of chromosomal imbalances in entire genomes. The CGH approach is used in cancer research to identify over- and under-representations of chromosomal regions. To search for and analyze tumor-relevant aberration patterns in CGH data, we designed, implemented, and deployed a relational database system. This project is part of a more complex and comprehensive effort to compile, integrate, fuse, and analyze biological and clinical data from heterogeneous and distributed information sources. In this article, we discuss the obstacles and pitfalls that were encountered in the design process, describe the resulting CGH database model and the underlying technical infrastructure, and present the first results based on mining the CGH database.

Detection of chromosome imbalances in retinoblastoma by parallel karyotype and CGH analyses

Detection of chromosome imbalances in retinoblastoma by parallel karyotype and CGH analyses

Detection of chromosomal imbalances in central neurocytomas by using comparative genomic hybridization

Central neurocytomas are rare neuronal tumors commonly found in the intraventricular regions. Little is known about the tumorigenesis of these neoplasms. The aim of this study was to provide an overview of genetic imbalances in central neurocytomas. In this study, comparative genomic hybridization was used to identify DNA sequence copy number changes (losses and gains) in a series of 10 central neurocytomas. Tumor DNA and normal reference DNA were differentially labeled and allowed to cohybridize to normal metaphase chromosomes. After hybridization and fluorescent staining of the bound DNA, regions of gain or of loss of DNA sequences were detected as changes in the tumor/normal fluorescence intensity ratio along the target metaphase chromosomes. A gain of DNA sequence was detected in chromosomes 2p, 10q, and 18q. A protooncogene, Bcl2, which maps to 18q21, was evaluated by immunohistochemical analysis to determine its role in the formation of central neurocytomas. In this study the authors identified recurrent genetic changes on chromosomes 2p, 10q, and 18q in central neurocytomas and highlighted chromosomal regions for additional mapping and cloning of candidate genes that are important in the development of central neurocytomas.

Detection of chromosomal imbalances in leiomyosarcoma by comparative genomic hybridization and interphase cytogenetics

Leiomyosarcomas comprise a group of malignant soft-tissue tumors with smooth-muscle differentiation. In this study, 14 cases of leiomyosarcoma were screened for changes in relative chromosome copy number by comparative genomic hybridization. A high number of imbalances (mean, 16.3; range, 6–26) was detected, with chromosomal gains occurring about twice as much as losses. The most frequent gains were found in 5p15, 8q24, 15q25→q26, 17p, and Xp (43% to 50%), whereas the most frequent losses were found in 10q and 13q (50% and 78%, respectively). Twenty high-level amplifications affecting 15 different chromosomal subregions were detected in nine different tumors. In three leiomyosarcomas, sequences on chromosome arm 17p were found to be highly amplified, with a minimal overlapping region on subbands 17p12→p11. We further discovered that the Smith-Magenis syndrome critical region on 17p11.2 is included in the 17p amplicons of two leiomyosarcoma cases. Using probes flanking this genetically unstable region, a mean of 14 and 22 signals per nucleus, respectively, was detected in both leiomyosarcomas by fluorescence in situ hybridization. In conclusion, this analysis identifies a number of characteristic chromosomal imbalances in leiomyosarcomas and provides evidence for the localization of potential oncogenes and tumor suppressor genes active in leiomyosarcoma genomes.

Detection of chromosome imbalances in retinoblastoma by parallel karyotype and CGH analyses

We have studied a series of 20 primary retinoblastomas by karyotypic analysis and comparative genomic hybridization (CGH), to perform an exhaustive evaluation of chromosome imbalances in this tumor. In addition, 4 tumors were studied by CGH only. On the whole, CGH results were largely in agreement with those of karyotypic analysis and with known cytogenetic data. The most frequent imbalances were +6p (13/24 cases), +1q (12/24), -16/-16q (11/24), and +2p (9/24). Recurrent high-level amplifications were observed in 2p23-25 and 1q21. Amplification of 2p23-25, present in 4 cases among which 3 showed double-minute chromosomes, was related to MYCN amplification, as demonstrated by FISH and PCR. No evident correlation was found in this small series between any of the imbalances identified and either the differentiation or the histoprognostic risk.

Detection of chromosomal imbalances in growth hormone-secreting pituitary tumors by comparative genomic hybridization

Although recent molecular investigations have identified a number of genetic alterations that are associated with the development of pituitary adenomas, the exact pathogenesis mechanism of these tumors remains largely unknown. In this study, we used a genome-wide survey to detect specific genetic changes within the genome of pituitary adenomas. A series of 10 growth hormone-secreting adenomas were analyzed for their genetic imbalances on all 22 autosomes by comparative genomic hybridization (CGH). Chromosomal imbalances were detected in 8 GH-secreting adenomas, whereas 2 tumors had no detectable genetic abnormalities. Chromosome gains were more frequent than losses. Overrepresentation of whole or parts of chromosomes were detected in 5 10 (50%) in 19, 3 10 (30%) in each of 5, 9, and 22q, 2 10 (20%) in 17p12-q21, whereas DNA loss were 3 10 (30%) in 13q and 2 10 (20%) in 18. No detectable gain or loss of genetic material was observed in chromosomes 7, 8, 10, 12, 15, and 20. The findings of overrepresentation of chromosomes 5q, 9p, 17q and DNA loss of chromosome 18 were consistent with those detected in nonfunctioning adenomas (Daniely M, Aviram A, Adams EF, et al: J Clin Endocrinol Metab 83:1801–1805, 1998) suggesting that the development of pituitary tumors, at least in somatotroph and nonfunctioning adenomas, may share common pathway. Frequent amplifications in chromosomes 19 and 22q imply that candidate genes residing in these chromosomal regions may be involved in the pathogenesis of GH-secreting adenomas.

Comparative genomic hybridization is a powerful tool, complementary to cytogenetics, to identify chromosomal abnormalities in childhood acute lymphoblastic leukaemia.

Cytogenetics has a strong prognostic value in childhood acute lymphoblastic leukaemia (ALL), but results are often incomplete because of the poor chromosome morphology. To improve this analysis, we tested comparative genomic hybridization (CGH) for the detection of chromosomal imbalances. 72 children were retrospectively analysed using CGH. Only 53% of the patients had been fully banded by standard methods. With CGH, 36 patients retained a normal chromosomal profile and 36 had unbalanced abnormalities. No amplification was detected. Fluorescence in situ hybridization (FISH) with centromeric and unique sequence probes was used in those cases with discrepancies or unsuccessful karyotype to validate CGH results. CGH enabled clear identification of unbalanced chromosomal abnormalities, even in some cases which had a normal karyotype. In view of the strong prognostic value of hyperdiploidy in childhood ALL, CGH appears to be a powerful technique, complementary to conventional cytogenetics.

Comparative genomic hybridization analysis of human neuroblastomas: Detection of distal 1p deletions and further molecular genetic characterization of neuroblastoma cell lines

Deletions of the short arm of chromosome 1 and MYCN amplification are the most frequently encountered genetic changes in disseminated neuroblastomas and neuroblastoma cell lines. Different strategies have been followed for detection of these and other genomic changes in neuroblastoma including karyotyping, FISH, and LOH, each with its own limitations. Here we report upon the evaluation of comparative genomic hybridization (CGH) in the analysis of neuroblastoma cell lines, with the emphasis on the assessment of the reliability of CGH for the detection of distal 1p deletions. We have analyzed seven neuroblastoma cell lines for which the 1p status was previously studied in detail using FISH and LOH. Our results show that CGH allows reliable detection of distal 1p deletions, including a small interstitial deletion in cell line SK-N-AS. Furthermore, CGH also allows the detection of chromosomal imbalances which would otherwise remain undetected, and provides useful information for further molecular characterization of chromosomal imbalances.