Conventional Comparative Genomic Hybridization Research Articles

Intellectual disability; an example of not relying on karyotype or array comparative genomic hybridization alone

Intellectual disability (ID) is a heterogeneous disorder which could have different etiologies. Among these etiologies, genetic causes are important issues. Genetic and pediatric guidelines have suggested using comparative genomic hybridization (CGH) as the first tire test for the diagnosis of such patients. However, in some ID cases such as our patient, we have demonstrated that considering both CGH array and karyotyping without any superiority would be beneficial. In the present report, we demonstrated an ID patient with rare chromosomal abnormalities reported in both conventional karyotype and CGH array.

Runs of homozygosity in spontaneous abortions from families with recurrent pregnancy loss

Recurrent pregnancy loss (RPL) is a severe reproductive pathology with a significant component of unexplained etiology. Extended homozygous regions as a possible etiological factor for RPL were sought in the genomes of embryos. Twenty-two paired first-trimester spontaneously aborted embryos from eleven women with recurrent miscarriage were analyzed. All embryos had normal karyotypes according to metaphase karyotyping and conventional comparative genomic hybridization. SurePrint G3 Human CGH + SNP 4 × 180K microarrays (Agilent Technologies) were used to search for homozygous regions. As a result, 39 runs of homozygosity (ROH) were identified in extraembryonic tissues of 15 abortuses. Verification of recurrent homozygous regions was performed by Sanger sequencing. The presence of occasional heterozygous SNPs was shown in 25 extended ROHs, which may indicate that they did not arise de novo but were inherited from parents. In the course of inheritance in a series of generations, they may accumulate mutations, leading to heterozygosity for several sites in the initially homozygous population-specific regions. Homozygotization of recessive mutations is one of the putative mechanisms of the influence of such inherited ROHs on RPL development. The high frequency of extended ROHs detected in the present study may point to a role of inbreeding in RPL etiology. Homozygous regions may also occur due to uniparental disomy, and abnormalities of genomic imprinting may be another mechanism responsible for the pathological manifestation of ROHs in embryogenesis. Indeed, five predicted imprinted genes were identified within ROHs according to the Geneimprint database: OBSCN, HIST3H2BB, LMX1B, CELF4, and FAM59A. This work reports the first finding of a high frequency of extended ROHs in spontaneously aborted embryos with normal karyotypes from families with RPL.

Comparative Genomic Hybridization (CGH) in Genotoxicology.

In the past two decades, comparative genomic hybridization (CGH) and array CGH have become indispensable tools in clinical diagnostics and toxicological risk assessment. Initially developed for the genome-wide screening of chromosomal imbalances, that is, copy-number variations in tumor cells, both CGH and array CGH have been employed in genotoxicology and most recently in toxicogenomics. The latter allows a multi-end point analysis of how particular genes react to toxic agents, revealing changes in signaling pathways and other underlying molecular mechanisms. This chapter provides background on the use of CGH and array CGH in the context of genotoxicology, and also a protocol for conventional CGH, so that the basic principles of this methodology can be better understood. Conventional and array CGH investigate DNA expression patterns, copy-number variations across the whole genome, and loss of heterozygosity after genotoxic damage. Array CGH is still cost-intensive but produces exponentially more data, requiring suitable analytical algorithms and sophisticated bioinformatic analysis. As toxicogenomics is an emerging sub-discipline of toxicology research, effectively evaluating toxicogenomic microarray data can be hugely advantageous for human risk assessment, even though international regulatory guidelines on toxicogenomics have yet to be fully agreed and implemented.

Comparative Genomic Hybridization (CGH) for Prenatal Diagnosis of Wolf-Hirschhorn Syndrome

Wolf-Hirschhorn Syndrome (WHS) is a rare genetic condition caused by partial deletion of the short arm of chromosome 4 (4p-). Variability in 4p deletions and rearrangements leads to a wide spectrum of clinical manifestations of this disease. Most prenatal WHS diagnoses are associated with large 4p deletions identified by conventional cytogenetic techniques; however some submicroscopic deletions can only be diagnosed using molecular techniques. In this case report, a combined diagnostic approach based on conventional karyotyping and comparative genomic hybridization (CGH) offered a quick and definitive result to allow accurate prognoses and genetic counseling of WHS for the family.

Vaccine strain varicella-zoster virus–induced central nervous system vasculopathy as the presenting feature of DOCK8 deficiency

Vaccine strain varicella-zoster virus–induced central nervous system vasculopathy as the presenting feature of DOCK8 deficiency

Chromosomal aberrations in primary PDGFRA-mutated gastrointestinal stromal tumors

Approximately 15% of gastrointestinal stromal tumors (GISTs) harbor mutations in the platelet-derived growth factor receptor α (PDGFRA) gene. Chromosomal aberrations play a crucial role in tumor progression and correlate with clinical behavior. Imbalances, particularly in PDGFRA-mutated GISTs, have not yet been evaluated in larger series. We analyzed 53 PDGFRA-mutated GISTs (including 2 with corresponding metastases) for chromosomal imbalances by conventional comparative genomic hybridization and compared them with a historical collective of 122 KIT-mutated GISTs. PDGFRA exon 18 mutations (91% of cases) and exon 12 mutations (9% of cases) correlated significantly with gastric and intestinal sites, respectively. The most common aberrations were identical to those found in KIT-mutated GISTs, with -14q in 70%, -1p in 28%, and -22q in 17% of cases. Overall, there were significantly fewer chromosomal aberrations compared with KIT-mutated GISTs, with a mean of 2.8 (0.6 gains, 2.1 losses) aberrations per tumor. There was a statistically significant association of more than 5 chromosomal imbalances with intermediate/high-risk categories. Regarding specific chromosomal aberrations, -9p, -13q, and -22q correlated with intermediate/high risk, and -1p and +8q with poorer survival, although progression occurred in only 2 cases. Altogether, PDGFRA-mutated GISTs display the same chromosomal aberrations as KIT-mutated GISTs, although they have a lower degree of chromosomal instability in line with their generally favorable outcome.

Amplification of the STOML3, FREM2, and LHFP Genes Is Associated with Mesenchymal Differentiation in Gliosarcoma

Gliosarcoma is a rare glioblastoma variant characterized by a biphasic tissue pattern with alternating areas that display either glial (glial fibrillary acidic protein-positive) or mesenchymal (reticulin-positive) differentiation. Previous analyses have shown identical genetic alterations in glial and mesenchymal tumor areas, suggesting that gliosarcomas are genetically monoclonal, and mesenchymal differentiation was considered to reflect the elevated genomic instability of glioblastomas. In the present study, we compared genome-wide chromosomal imbalances using array comparative genomic hybridization in glial and mesenchymal tumor areas of 13 gliosarcomas. The patterns of gain and loss were similar, except that the gain at 13q13.3-q14.1 (log(2) ratio >3.0), containing the STOML3, FREM2, and LHFP genes, which was restricted to the mesenchymal tumor area of a gliosarcoma. Further analyses of 64 cases of gliosarcoma using quantitative PCR showed amplification of the STOML3, FREM2, and LHFP genes in 14 (22%), 10 (16%), and 7 (11%) mesenchymal tumor areas, respectively, but not in glial tumor areas. Results of IHC analysis confirmed that overexpression of STOML3 and FREM2 was more extensive in mesenchymal than in glial tumor areas. These results suggest that the mesenchymal components in a small fraction of gliosarcomas may be derived from glial cells with additional genetic alterations.

DNA Copy Number Changes and Immunophenotype Pattern in Karyotypically Normal Acute Myeloid Leukemia Patients from an Indian Population

Chromosomal abnormalities are important in the diagnosis and prognosis of patients with acute myeloid leukemia (AML). The purpose of this study was to identify DNA copy number variations in karyotypically normal AML patients and their correlation with immunophenotypes. Conventional comparative genomic hybridization (CGH) and immunophenotyping were performed in 46 untreated AML patients aged 7-68 years. Among the 86 Indian patients who had AML, 40 (46.5%) showed an abnormal karyotype and 46 (53.4%) showed no chromosome aberrations. The karyotypically abnormal AML patients were excluded from the study. Out of the 46 patients without chromosomal aberrations, 24 (52.2%) showed DNA copy number variations including losses and gains. The DNA copy number variations involved chromosomes 1, 3, 6, 12, 15, 16, 17 (gains) and 1, 4, 2, 3, 5, 7, 8, 9, 10, 11, 13, 15, 18, 20, 21 (losses). The aberrant immunophenotype was noticed in 13 of these 24 (54%) cases. The hidden chromosome rearrangements in karyotypically normal AML, which could not be detected by conventional cytogenetics and fluorescence in situ hybridization, were detected by CGH. These genetic changes have an important role in the prognosis of the disease. The DNA copy number changes might also be involved in the aberrant immunophenotypes in our study.

Genotypic and Phenotypic Differences between Nodal and Extranodal Diffuse Large B-Cell Lymphomas

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group of diseases that have diverse clinical, pathological, and biological features. Here, it is shown that primary nodal and extranodal DLBCLs differ genomically and phenotypically. Using conventional comparative genomic hybridization (CGH), the authors assessed the chromosomal aberrations in 18 nodal, 13 extranodal, and 5 mixed DLBCLs. The results demonstrate significantly distinct chromosomal aberrations exemplified by gains of chromosomal arms 1p, 7p, 12q24.21-12q24.31, and 22q and chromosome X and loss of chromosome 4, 6q, and 18q22.3-23 in extranodal compared with nodal DLBCLs. Nodal DLBCLs showed an increased tendency for 18q amplification and BCL2 protein overexpression compared with extranodal and mixed tumors. Using a panel of five antibodies against GCET1, MUM1, CD10, BCL6, and FOXP1 proteins to subclassify DLBCLs according to the recent Choi algorithm, the authors showed that the genomic profiles observed between the nodal and extranodal DLBCLs were not due to the different proportions of GCB vs ABC in the two groups. Further delineation of these genomic differences was illuminated by the use of high-resolution 21K BAC array CGH performed on 12 independent new cases of extranodal DLBCL. The authors demonstrated for the first time a novel genome and proteome-based signatures that may differentiate the two lymphoma types.

Comprehensive embryo analysis of advanced maternal age–related aneuploidies and mosaicism by short comparative genomic hybridization

The short comparative genomic hybridization (short-CGH) method was used to perform a comprehensive cytogenetic study of isolated blastomeres from advanced maternal age embryos, discarded after fluorescent in situ hybridization (FISH) preimplantation genetic screening (PGS), detecting aneuploidies (38.5% of which corresponded to chromosomes not screened by 9-chromosome FISH), structural aberrations (31.8%), and mosaicism (77.3%). The short-CGH method was subsequently applied in one PGS, achieving a twin pregnancy.

Rates of loss of heterozygosity and mitotic recombination in NF2 schwannomas, sporadic vestibular schwannomas and schwannomatosis schwannomas

Biallelic inactivation of the NF2 gene occurs in the majority of schwannomas. This usually involves a combination of a point mutation or multiexon deletion, in conjunction with either a second point mutation or loss of heterozygosity (LOH). We have performed DNA sequence and dosage analysis of the NF2 gene in a panel of 239 schwannoma tumours: 97 neurofibromatosis type 2 (NF2)-related schwannomas, 104 sporadic vestibular schwannomas (VS) and 38 schwannomatosis-related schwannomas. In total, we identified germline NF2 mutations in 86 out of 97 (89%) NF2 patients and a second mutational event in 77 out of 97 (79%). LOH was by far the most common form of second hit. A combination of microsatellite analysis with either conventional comparative genomic hybridization (CGH) or multiplex ligation-dependent probe amplification (MLPA) identified mitotic recombination (MR) as the cause of LOH in 14 out of 72 (19%) total evaluable tumours. Among sporadic VS, at least one NF2 mutation was identified by sequence analysis or MLPA in 65 out of 98 (66%) tumours. LOH occurred in 54 out of 96 (56%) evaluable tumours, but MR only accounted for 5 out of 77 (6%) tested. LOH was present in 28 out of 34 (82%) schwannomatosis-related schwannomas. In all eight patients who had previously tested positive for a germline SMARCB1 mutation, this involved loss of the whole, or part of the long arm, of chromosome 22. In contrast, 5 out of 22 (23%) tumours from patients with no germline SMARCB1 mutation exhibited MR. High-resolution Affymetrix SNP6 genotyping and copy number (CN) analysis (Affymetrix, Santa Clara, CA, USA) were used to determine the chromosomal breakpoint locations in tumours with MR. A range of unique recombination sites, spanning approximately 11.4 Mb, were identified. This study shows that MR is a mechanism of LOH in NF2 and SMARCB1-negative schwannomatosis-related schwannomas, occurring less frequently in sporadic VS. We found no evidence of MR in SMARCB1-positive schwannomatosis, suggesting that susceptibility to MR varies according to the disease context.

Identification of genetic alterations in upper urinary tract urothelial carcinoma in end‐stage renal disease patients

Clinical presentations of end-stage renal disease (ESRD) patients on dialysis with upper urinary tract urothelial carcinoma (UUT-UC) are different from those with normal renal function. The pathogenesis remains unknown. We investigated the pathogenetic influence of chromosomal aberrations in patient on dialysis with UUT-UC. The chromosomal aberrations of UUT-UC specimens from seven dialysis patients were assessed by conventional comparative genomic hybridization (cCGH). Subsequently, we further investigated 20 cases by whole genome and fine-tiling oligonucleotide array-based CGH to demonstrate gains and losses, and compared with the clinicopathologic background. The chromosomal aberrations in UUT-UC specimens from dialysis patients were more complex than in bladder urothelial carcinoma (B-UC). Our data showed that gains at 5p, 7, 19q, and losses at 4q, 9p, and 15q are common in UUT-UC of ESRD patients. Gains in regions associated with DNA repair genes were noted in this study. High-stage and high-grade tumors displayed more copy number variants. In addition, female ESRD patients with UUT-UC had more frequent chromosomal aberrations than their male counterparts. In conclusion, unique chromosomal aberrations were indentified in UUT-UC in ESRD patients.

The Use of Comparative Genomic Hybridization and Fluorescent in Situ Hybridization in Postmortem Pathology Investigation of Congenital Malformations

Chromosomal abnormalities are an important cause of multiple congenital anomalies (MCA). However, conventional cytogenetic analysis using culture is unsuccessful in 10% to 40% of the cases. The purpose of this study was to examine if retrospective chromosomal analysis was possible on paraffin-embedded autopsy material with new techniques, including comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH). We investigated 92 patients, including 71 patients with MCA, 17 patients with an isolated congenital anomaly, and 4 normal controls, by conventional CGH analysis and/or FISH. The karyotype was known in 52 cases, of which 26 patients were normal and 26 had chromosomal anomalies. Comparative genomic hybridization or FISH confirmed all but 2 cases, which were not interpretable. In 40 patients the karyotype was unknown but could be analyzed successfully in 36 cases (90%) by CGH. In this series, we found 1 additional chromosomal aberration, 45,X (Turner syndrome). Furthermore, we examined the postmortem material of 12 patients by FISH, confirming a known abnormal karyotype in 9 patients, an abnormal karyotype found by CGH in 1 case, and confirming DiGeorge syndrome (22q11 deletion) in twins. Comparative genomic hybridization and FISH are reliable techniques with which to perform retrospective genetic analysis on paraffin-embedded autopsy material.

An efficient protocol for the detection of chromosomal abnormalities in spontaneous miscarriages or foetal deaths

Objective Characterization of chromosomal abnormalities in 232 spontaneous miscarriages or foetal deaths using both classical and molecular cytogenetics. Study design Chromosomal abnormalities are responsible for 40–50% of all early pregnancy losses. Conventional cytogenetics is associated with 10–40% of culture failure. Comparative genomic hybridization (CGH) is a DNA-based technique that screens chromosome imbalances in the whole genome and may overcome this problem, although additional methods are required to distinguish between different ploidies, mosaicisms and maternal cell contamination. For a full characterization of chromosomal aberrations in 232 spontaneous miscarriages or foetal deaths we applied a sequential protocol that uses conventional cytogenetics, plus CGH and touch fluorescence in situ hybridization (Touch FISH). Results Successful karyotyping was obtained in 173/232 (74.6%) of the cases, 66/173 (38.2%) of which had an abnormal chromosomal complement. CGH and Touch FISH analyses revealed another 19 abnormal cases in the 63 failures of culture. Overall there were 85/233 (36.6%) cases with an abnormal chromosomal complement, with examples from all three trimesters. Comparing cases, with or without chromosomal abnormalities, no statistical differences were found between women with one or recurrent miscarriages. On the contrary, significant differences were found comparing mean maternal ages or mean gestational ages, in cases with or without chromosomes abnormalities. Conclusion Adopting this sequential protocol, chromosomal complement information was available even in cases with culture failure.

Comparative Genomic Hybridization Analysis Reveals New Different Subgroups in Early-stage Bladder Tumors

To classify bladder tumors according to their genomic imbalances and evaluate their association with patient's outcome. Sixty-three superficially and minimally invasive bladder tumors were analyzed by conventional comparative genomic hybridization. Subtelomeric screening in 15 of these tumors was performed by multiplex ligation-dependent probe amplification. Losses of 9q and 9p (32% and 25% of all cases, respectively) as well as gains of chromosomes Xq and Xp (28% and 25%, respectively) were the most frequent chromosome imbalances. Losses of 8p and gains in 1q and 8q were detected in >20% of cases. Tumors were classified into 3 groups according to their individualized pattern of gains and losses. The largest group was characterized by few chromosome imbalances, presenting 77% and 49% of the Ta and T1 tumors, respectively. Another group characterized by chromosomal gains, was composed of equal number of Ta and T1 tumors, with +1q and +17q gains being the most common imbalances. A minority group was characterized by chromosomal losses on 11q, 5q, and 6q. The multiplex ligation-dependent probe amplification study showed good correlation with comparative genomic hybridization results. With regard to the biological significance of this classification, a remarkable fact is that this minority group composed mainly of T1 tumors, showed a significant decrease in patient overall survival. Our data suggest that superficial carcinomas of the bladder can be subdivided into a larger number of subclasses than had previously been expected. Our results also demonstrate a decreased survival among patients whose tumors show more genomic losses than gains.

Genetic alterations detected by comparative genomic hybridization and recurrence rate in epithelial ovarian carcinoma

To assess the putative correlation between comparative genomic hybridization (CGH)-detectable genetic alterations in epithelial ovarian cancer and disease recurrence, conventional CGH was performed on 45 epithelial ovarian cancers: 26 tumors from sporadic, BRCA mutation noncarriers and 11 and 8 tumors from BRCA1 and BRCA2 mutation carriers, respectively. Relevant clinical data, including histology, grade, stage, size of residual tumor, recurrence, and survival, were obtained from outpatient and inpatient charts. Among the 45 cases, the most common regions involving gain of DNA copy number were 3q ( n = 23; 51%), 8q ( n = 21; 47%), and 1q ( n = 14; 31%), and the most common regions with loss were 19 and 22 at 9 cases (20%) each, followed by 5q ( n = 6; 13%). In multivariate analysis, the total number of genetic alterations was not associated with risk of recurrence, but gain in 5p was associated with a higher risk of recurrence (hazard ratio HR = 6.06, P = 0.0399), and gain in 1p as well as loss in 5q were associated with a significant decrease in recurrence (HR = 0.08, P = 0.0079, and HR = 0.10, P = 0.0143, respectively). Recurrence rate in patients with epithelial ovarian cancer is seemingly associated with specific genetic alterations detected by CGH, but the specific genes involved and the implications of these findings await further studies.

Epithelioid trophoblastic tumor: comparative genomic hybridization and diagnostic DNA genotyping

Arising from the putative chorionic-type intermediate trophoblast, epithelioid trophoblastic tumor is a recent addition to the spectrum of gestational trophoblastic diseases. Frequently, the tumor involves the uterine cervix and is misdiagnosed as invasive squamous-cell carcinoma. The pathogenesis of the tumor is poorly understood, and its molecular analysis is essentially lacking. This study was designed to explore chromosomal alterations in epithelioid trophoblastic tumor and to use DNA genotyping to demonstrate its trophoblastic origin, therefore separating the tumor from its mimics of the maternal origin. Five cases of epithelioid trophoblastic tumors were included in this study and paired DNA samples from the tumor and normal tissue were extracted from paraffin-embedded archival materials. The status of chromosomal alterations was analyzed by comparative genomic hybridization using conventional metaphase chromosome preparations. The parental genetic contribution was determined by DNA genotyping analysis using AmpFISTR® Identifiler™ Amplification system (Applied Biosystems Inc.). Comparative genomic hybridization analysis was successful in three cases analyzed, all of which showed a balanced chromosomal profile without detectable gain or loss of the genome. DNA genotyping was informative in four epithelioid trophoblastic tumor involving anatomic locations including the cervix (two cases), endomyometrium (one case) and lung (metastatic, one case). All four cases were found to have unique paternal alleles, confirming the trophoblastic nature of the tumors. In summary, chromosomal alterations detectable by conventional comparative genomic hybridization are not features of epithelioid trophoblastic tumors. In difficult cases, the presence of the paternal alleles demonstrated by DNA genotyping is a powerful diagnostic application in separating an epithelioid trophoblastic tumor from its maternal mimics, particularly the far more common squamous-cell carcinoma of the uterine cervix.

Array-comparative genomic hybridization in sporadic benign pheochromocytomas

Pheochromocytomas (PCC) are catecholamine-producing tumors arising from the adrenal medulla that occur either sporadically or in the context of hereditary cancer syndromes, such as multiple endocrine neoplasia type 2 (MEN2), von Hippel-Lindau disease (VHL), neurofibromatosis type 1, and the PCC-paraganglioma syndrome. Conventional comparative genomic hybridization studies have shown loss of 1p and 3q in the majority of sporadic and MEN2-related PCC, and 3p and 11p loss in VHL-related PCC. The development of a submegabase tiling resolution array enabled us to perform a genome-wide high-resolution analysis of 36 sporadic benign PCC. The results show that there are two distinct patterns of abnormalities in these sporadic PCC, one consisting of loss of 1p with or without concomitant 3q loss in 20/36 cases (56%), the other characterized by loss of 3p with or without concomitant 11p loss in 11/36 (31%). In addition, we found loss of chromosome 22q at high frequency (35%), as well as the novel finding of high frequency chromosome 21q loss (21%). We conclude that there appear to be two subgroups of benign sporadic PCC, one of which has a pattern of chromosomal abnormalities that is comparable with PCC from patients with MEN2 and the other that is comparable with the PCC that arise in patients with VHL disease. In addition, genes on 21q and 22q might play a more important role in PCC pathogenesis than had been assumed thus far.

Prediction of S-1-induced anemia

S-1, a novel oral fluoropyrimidine, has shown remarkably good tolerability in Korean gastric and colorectal cancer patients due to its favorable safety profile. Myelosuppression and diarrhea were the events that precluded dose escalation in Japan, whereas gastrointestinal toxicity and skin reaction were the major limiting factors in Western countries. In contrast, the major adverse event in Korean patients was anemia, which appeared early in the S-1 treatment period and varied among patients. Conventional comparative genomic hybridization (CGH) is used to screen for chromosomal copy number variations such as gene gain, loss, amplification, and deletion. This technique can provide information about genetic instability and chromosomal rearrangements. However, the low resolution of 5–10 Mb is a caveat with conventional CGH. cDNA microarray-based CGH is a useful technique for achieving higher resolution for the detection of genomic aberrations. Pharmacogenomic markers, in combination with clinical factors such as initial hemoglobin level, may be useful for predicting S-1 treatment-induced anemia. Prospective genomic and clinical validation of this model may provide a predictive model for the clinical application of S-1 treatment.

Single-cell isolation from cell suspensions and whole genome amplification from single cells to provide templates for CGH analysis

A comprehensive genomic analysis of single cells is instrumental for numerous applications in tumor genetics, clinical diagnostics and forensic analyses. Here, we provide a protocol for single-cell isolation and whole genome amplification, which includes the following stages: preparation of single-cell suspensions from blood or bone marrow samples and cancer cell lines; their characterization on the basis of morphology, interphase fluorescent in situ hybridization pattern and antibody staining; isolation of single cells by either laser microdissection or micromanipulation; and unbiased amplification of single-cell genomes by either linker-adaptor PCR or GenomePlex library technology. This protocol provides a suitable template to screen for chromosomal copy number changes by conventional comparative genomic hybridization (CGH) or array CGH. Expected results include the generation of several micrograms of DNA from single cells, which can be used for CGH or other analyses, such as sequencing. Using linker-adaptor PCR or GenomePlex library technology, the protocol takes 72 or 30 h, respectively.