Minimal sizes of deletions detected by comparative genomic hybridization

Comparative genomic hybridization (CGH) has been used widely for the molecular cytogenetic analysis of tumors. Until now, the spatial resolution of this technique for diagnosing deletions of chromosomal sequences has not been assessed in detail. In the present study, we performed CGH analyses on five DNA samples derived from B-cell leukemias with 11q deletions, the sizes of which ranged from 3 Mbp to 14–18 Mbp. CGH experiments were evaluated by two established commercial analysis systems. Deletions down to a size of 10–12 Mbp were diagnosed based on a diagnostic threshold value of 0.8, if the vast majority of cells carried the deletion. For cases with smaller deletions, the ratio profiles were shifted toward underrepresentation at the respective chromosomal bands; however, the diagnostic threshold value was not reached. In all five cases, there was complete agreement between the two image analysis systems. Genes Chromosomes Cancer 21:172–175, 1998. © 1998 Wiley-Liss, Inc.     

Fine-mapping subtelomeric deletions and duplications by comparative genomic hybridization in 42 individuals

Human subtelomere regions contain numerous gene-rich segments and are susceptible to germline rearrangements. The availability of diagnostic test kits to detect subtelomeric rearrangements has resulted in the diagnosis of numerous abnormalities with clinical implications including congenital heart abnormalities and mental retardation. Several of these have been described as clinically recognizable syndromes (e.g., deletion of 1p, 3p, 5q, 6p, 9q, and 22q). Given this, fine-mapping of subtelomeric breakpoints is of increasing importance to the assessment of genotype-phenotype correlations in these recognized syndromes as well as to the identification of additional syndromes. We developed a BAC and cosmid-based DNA array (TEL array) with high-resolution coverage of 10 Mb-sized subtelomeric regions, and used it to analyze 42 samples from unrelated patients with subtelomeric rearrangements whose breakpoints were previously either unmapped or mapped at a lower resolution than that achievable with the TEL array. Six apparently recurrent subtelomeric breakpoint loci were localized to genomic regions containing segmental duplication, copy number variation, and sequence gaps. Small (1 Mb or less) candidate gene regions for clinical phenotypes in separate patients were identified for 3p, 6q, 9q, and 10p deletions as well as for a 19q duplication. In addition to fine-mapping nearly all of the expected breakpoints, several previously unidentified rearrangements were detected.     

1p and 3p deletions in meningiomas without detectable aberrations of chromosome 22 identified by comparative genomic hybridization

Meningioma is a common tumor of the meninges covering the central nervous system. Although generally a benign tumor, meningioma often recurs and is malignant in 5–10% of all cases. Loss of chromosome 22 loci, and specifically inactivation of the NF2 tumor suppressor gene, is considered one of several critical steps in the tumorigenesis of meningioma. However, cytogenetic and molecular investigations have failed to detect either aberrations of chromosome 22 or mutations in the NF2 gene in approximately 40% of all tumors, thus making it apparent that an alternative mechanism(s) is responsible for the development of a large fraction of meningiomas. This subset of meningiomas is not distinct with regard to clinical and histopathological features from tumors showing deletions on chromosome 22. It is, therefore, important to attempt the elucidation of molecular pathway(s) that may operate in the tumorigenesis of these tumors. We used comparative genomic hybridization (CGH) to identify regions of the genome other than chromosome 22, contributing to the development of meningioma. We analyzed 25 tumors that had undergone detailed LOH analysis on chromosome 22 and were shown to contain no detectable deletions. Two benign, malignancy grade I, meningiomas showed concurrent deletion of 1p and 3p. These results suggest that loss of both 1p and 3p may contribute to meningioma tumorigenesis. This may represent genetic changes that are alternative to deletions on chromosome 22. Genes Chromosomes Cancer 20:419–424, 1997. © 1997 Wiley-Liss, Inc.     

Array-based comparative genomic hybridization analysis of recurrent chromosome 15q rearrangements

Genomic rearrangements of chromosome 15q11-q13 cause diverse phenotypes including autism, Prader-Willi syndrome (PWS), and Angelman syndrome (AS). This region is subject to genomic imprinting and characterized by complex combinations of low copy repeat elements. Prader-Willi and Angelman syndrome are caused primarily by 15q11-13 deletions of paternal and maternal origin, respectively. Autism is seen with maternal, but not paternal, interstitial duplications. Isodicentric 15q, most often of maternal origin, is associated with a complex phenotype often including autistic features. Limitations of conventional cytogenetic tests preclude a detailed analysis in most patients with 15q rearrangements. We have developed a microarray for comparative genomic hybridization utilizing 106 genomic clones from chromosome 15q to characterize this region. The array accurately localized all breakpoints associated with gains or losses on 15q. The results confirmed the location of the common breakpoints associated with interstitial deletions and duplications. The majority of idic(15q) chromosomes are comprised of symmetrical arms with four copies of the breakpoint 1 to breakpoint 5 region. Patients with less common breakpoints that are not distinguished by routine cytogenetic methods were more accurately characterized by array analysis. This microarray provides a detailed characterization for chromosomal abnormalities involving 15q11-q14 and is useful for more precise genotype-phenotype correlations for autism, PWS, AS, and idic(15) syndrome.     

Recurrent chromosome alterations in hepatocellular carcinoma detected by comparative genomic hybridization

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and has a very poor prognosis. Fifty primary HCC cases have been analyzed in the present study to explore the association between genomic alteration in primary HCC and clinical features. Several recurrent chromosomal abnormalities were identified in this study. The most frequently detected chromosomal gains involved chromosome arms 1q (33/50 cases, 66%), 8q (24/50 cases, 48%), and 20q (10/50 cases, 20%). High-copy-number amplifications involving 1q (4 cases), 8q (3 cases), and 20q (3 cases) were detected, and a minimum overlapping amplified region at 1q12-q22 was identified. The most frequently detected loss of chromosomal material involved 16q (35/50 cases, 70%), 17p (26/50 cases, 52%), 19p (21/50 cases, 42%), 4q (20/50 cases, 40%), 1p (18/50 cases, 36%), 8p (16/50 cases, 32%), and 22q (14/50 cases, 28%). The associations between genomic alterations detected in the present study and clinical features including clinical stage, tumor size, HBV infection, chronic liver disease, and liver cirrhosis were explored. Our CGH results suggest that the gain of 20q and deletion of 8p are late genetic alterations in HCC, because the incidence of these alterations was obviously increased in the advanced clinical stages. Another finding showed that loss of 8p and gain of 8q and 20q are associated with tumor size. The recurrent gain and loss of chromosomal regions identified in this study provide candidate regions that may contain oncogenes or tumor suppressor genes respectively involved in HCC development and progression.     

Preimplantation genetic diagnosis and chromosome analysis of blastomeres using comparative genomic hybridization

Numerical chromosome errors are known to be common in early human embryos and probably make a significant contribution to early pregnancy loss and implantation failure in IVF patients. Over recent years fluorescent in situ hybridization (FISH) has been used to document embryonic aneuploidies. Many IVF laboratories perform preimplantation genetic diagnosis (PGD) with FISH to select embryos that are free from some aneuploidies in an attempt to improve implantation, pregnancy and live birth rates in particular categories of IVF patients. The usefulness of FISH is limited because only a few chromosomes can be detected simultaneously in a single biopsied cell. Complete karyotyping at the single cell level can now be achieved by comparative genomic hybridization (CGH). CGH enables not only enumeration of all chromosomes but gives a more complete picture of the entire length of each chromosome and has demonstrated that chromosomal breakages and partial aneuploidies exist in embryos. CGH has provided invaluable information about the extent of mosaicism and aneuploidy of all chromosomes in early human conceptuses. CGH has been applied to clinical PGD and has resulted in the birth of healthy babies from embryos whose full karyotype was determined in the preimplantation phase.     

Mapping of chromosomal gains and losses in prostate cancer by comparative genomic hybridization

Comparative genomic hybridization (CGH) allows detection of chromosomal imbalances in whole genomes in a comprehensive manner. With this approach, ten cases of prostate cancer (seven primary tumors and three metastases) were analyzed. Frequent chromosomal gains detected by CGH involved chromosome arms 7q, 8q, 9q, and 16p, and chromosomes 20 and 22, as well as frequent losses of chromosome arms 16q and 18q, in at least three of the ten cases. Overrepresentation of chromosome arm 9q has not been described in published reports. The CGH data were compared with results of a loss of heterozygosity (LOH) study, in which complete allelotyping was performed in the same prostate tumors with 74 different polymorphic markers. In general, a high concordance between the CGH and LOH results was observed (92%). Tumors revealing discrepancies by CGH and LOH analysis were investigated further by interphase cytogenetics, and the resulting picture regarding the genomic alterations is discussed in detail.     

Characterization of chromosome aberrations associated with soft-tissue leiomyosarcomas by twenty-four-color karyotyping and comparative genomic hybridization analysis

Data on the chromosome aberrations associated with leiomyosarcomas of soft tissues are limited, complex, and incomplete. The aim of this study was to characterize genetic aberrations associated with this tumor group, to identify consistent regions of involvement and to determine correlations with clinical outcome. Chromosomes were prepared from 10 primary soft-tissue leiomyosarcoma samples, and preparations from four of them, plus the cell line SK-LMS-1, were suitable for analysis using 24-color karyotyping by multifluor fluorescence in situ hybridization. This method allowed rearranged chromosomes to be characterized, which would not have been possible by banding analysis alone. The remaining six chromosome preparations were analyzed using standard Giemsa banding. The chromosome imbalances associated with all the samples were determined by comparative genomic hybridization analysis. Taken together, the results show both intra- and intertumor heterogeneity and considerable complexity. Although no highly consistent rearrangements were found, some regions of the genome frequently were involved, including 1q21, 5p14-pter, and 20q13, which likely harbor genes that play a role in the pathogenesis of soft-tissue leiomyosarcomas. There were no obvious correlations between the chromosomal changes identified and available clinical details.     

Mapping of chromosomal gains and losses in primitive neuroectodermal tumors by comparative genomic hybridization

A series of 18 primitive neuroectodermal tumors (PNETs), the most common malignant central nervous system tumors of childhood, were analyzed with the recently developed approach of comparative genomic hybridization (CGH). In five cases, in which only small amounts of DNA were available, universal polymerase chain reaction was successfully applied to generate adequate probe material. In 15 tumors, chromosomal imbalances were elicited, most frequently involving chromosome 17 (loss of 17p and gain of 17q). Further recurrent imbalances included gains of the distal regions of 4p, 5p, 5q, 7q, 8q, and 9p. High-level amplifications were found on 2p24 (one case) and 8q24 (three cases), suggesting involvement of the protooncogenes MYCN and MYC, respectively. In one of these cases, Southern blot analysis could be performed, proving high-copy-number amplification of MYC. Interestingly, none of the three patients with high-copy-number amplifications of (MYC responded to therapy. Genes Chromosom Cancer 16:196–203 (1996). © 1996 Wiley-Liss, Inc.     

Comparative genomic hybridization reveals novel chromosome deletions in 90 primary soft tissue tumors

Uterine leiomyomas are the most common benign tumor that arise from smooth muscle cells of the myometrium. Little is known about the etiology and pathogenesis of this tumor. To investigate the molecular pathogenesis of these tumors, we have conducted an allelotype of 102 leiomyomas from 12 patients, using 67 fluorescently-tagged oligonucleotide primers amplifying microsatellite loci covering all autosomes. No areas of the genome showed frequent loss of heterozygosity (LOH); however, the highest rate of LOH (9%) was observed on 7q, consistent with previous cytogenetic observations. Uterine leiomyomas are sometimes multiple. In general, multiplicity of other types of neoplasm is associated with genetic predisposition to the disease. Because multiple tumors were available from each of the 12 patients studied, we looked for evidence of allele-specific LOH, which might indicate the presence of an underlying predisposition gene. However, no evidence for allele-specific LOH was detected, indicating that if cases of multiple uterine leiomyoma are due to an underlying predisposition gene, it is unlikely to be a recessive oncogene.     

Frequent gain of chromosome 19 in megakaryoblastic leukemias detected by comparative genomic hybridization

Acute megakaryocytic leukemia is a rare subtype of AML that is often difficult to diagnose; it is most commonly associated with Down syndrome in children. To identify chromosomal imbalances and rearrangements associated with acute megakaryocytic leukemia, we used G-banding, comparative genomic hybridization (CGH), and whole chromosome painting (WCP) on a variety of primary patients' samples and leukemia cell lines. The most common abnormality was gain of chromosome 19 or arm 19q, which was detected by CGH in four of 12 (33.3%) primary samples and nine of 11 (81.8%) cell lines. In none of the primary samples was this abnormality detected by G-banding analysis. WCP was used to define further the nature of the chromosome 19 gain in the cell lines, which was found to be due to the presence of additional 19q material on marker chromosomes or to cryptic translocations involving 19q. The most common chromosomal loss--detected only in the cell lines--was deletion of chromosomal band 13q14, which was seen in six of 11 (54.5%) cell lines. Other recurrent changes included gains of 1p, 6p, 8q, 11q, 15q, 17q, and 21q and losses of 2, 4q, 5q, 7q, 9p, and 11p. Combining conventional and molecular cytogenetic analyses defined recurrent clonal chromosomal abnormalities, which will aid in the identification of critical genes that are abnormal in acute megakaryocytic leukemia cells.     

Recurrent chromosome changes in 62 primary gastric carcinomas detected by comparative genomic hybridization

Comparative genomic hybridization (CGH) has been applied to detect recurrent chromosome alterations in 62 primary gastric carcinomas. Several nonrandom chromosomal changes, including gains of 8q (31 cases, 50%), 20q (29 cases, 47%) with a minimum gain region at 20q11. 2-q12, 13q (21 cases, 34%) with a minimum gain region at 13q22, and 3q (19 cases, 31%) were commonly observed. The regions most frequently lost included: 19p (23 cases, 37%), 17p (21 cases, 33%), and 1p (14 cases, 23%). High copy number gain (DNA sequence amplification) was detected in 6 cases. Amplification of 8q23-q24.2 and 20q11.2-q12 were observed in 3 cases. Gain of 20q and loss of 19p were confirmed by fluorescence in situ hybridization using corresponding bacterial artificial chromosomes (BAC) clones from those regions. The gain and loss of chromosomal regions identified in this study provide candidate regions involved in gastric tumorigenesis.     

乳腺叶状肿瘤染色体异常的比较基因组杂交分析初探

乳腺叶状肿瘤是一种较少见的乳腺肿瘤,近年来其发病率有逐渐增高的趋势。2003年WHO新分类中将其分为良性、交界性、恶性3种,其不同的病理类型反应了该瘤由良性发展为恶性的过程。我们应用比较基因组杂交（comparative genomic hybridization,CGH）技术分析乳腺叶状肿瘤不同类型染色体基因组DNA拷贝数的变化,初步探索该肿瘤的分子细胞遗传学变化,为进一步从全基因组染色体角度研究该瘤的恶变机制提供较有价值的资料。     

Array comparative genomic hybridization reveals a very high frequency of deletions of the long arm of chromosome 6 in testicular lymphoma

Despite the fact that numerous studies have been performed on diffuse large B-cell lymphoma (DLBCL), only few have concerned extranodal lymphomas occurring in the testis. We performed a cytogenetic and molecular study of 17 testicular non-Hodgkin lymphomas, of which 14 were proven primary DLBCL of the testis. Cytogenetic analysis revealed in 8 out of 11 evaluable cases a structural abnormality of the long arm of chromosome 6, with deletion or addition of material of unknown origin, and with breakpoints spanning the region 6q12-6q23. The cytogenetic findings were confirmed by fluorescent in situ hybridization (FISH) with a chromosome 6 painting probe. Using array based-comparative genomic hybridization on 16 evaluable cases, including 5 cases not tested by cytogenetics or FISH, 14 (88%) showed chromosome 6q deletions. We identified two regions of minimal deletion (RMD), at 104-113 Mb (6q16.3-q21) and 137.5-138.8 Mb (6q23.3), respectively. In one case, we observed a 2.7 Mb homozygous deletion ranging from 135.3 to 138.0 Mb that partly overlapped with the RMD at 6q23.3. Our study indicates that 6q deletions play a major pathogenetic role in DLBCL of the testis and that many of these deletions are part of unbalanced translocations.     

15q13q14 deletions: phenotypic characterization and molecular delineation by comparative genomic hybridization

We report on a detailed phenotypic characterization of two patients with novel de novo deletions involving 15q13q14, a chromosomal region immediately distal to the Prader-Willi/Angelman syndrome critical interval. Both cases were detected by the clinical array-based comparative genomic hybridization (array-CGH) and were precisely delineated through the high-density Agilent 244 K oligonucleotide array. The comparison of our patients with previously reported deletion cases involving the 15q13q14 region demonstrated a recurrent pattern of developmental anomalies including mild dysmorphic features, cleft palate/bifid uvula, congenital heart defects (PFO or ASD), developmental delay, and learning disabilities. The potential role of the genes within the deleted region in the pathogenesis of these various phenotypic abnormalities is discussed.     

Construction of a high-density and high-resolution human chromosome X array for comparative genomic hybridization analysis

The human chromosome X is closely associated with congenital disorders and mental retardation (MR), because it contains a significantly higher number of genes than estimated from the proportion in the human genome. We constructed a high-density and high-resolution human chromosome X array (X-tiling array) for comparative genomic hybridization (CGH). The array contains a total of 1,001 bacterial artificial chromosome (BACs) throughout chromosome X except pseudoautosomal regions and two BACs specific for Y. In four hybridizations using DNA samples from healthy males, the ratio of each spotted DNA was scattered between −3SD and 3SD, corresponding to a log₂ ratio of −0.35 and 0.35, respectively. Using DNA samples from patients with known congenital disorders, our X-tiling array was proven to discriminate one-copy losses and gains together with their physical sizes, and also to estimate the percentage of a mosaicism in a patient with mos 45,X[13]/46,X,r(X)[7]. Furthermore, array-CGH in a patient with atypical Schinzel-Giedion syndrome disclosed a 1.1-Mb duplication at Xq22.3 including a part of the IL1RAPL2 gene as a likely causative aberration. The results indicate our in-house X-tiling array to be useful for the identification of cryptic copy-number aberrations containing novel genes responsible for diseases such as congenital disorders and X-linked MR.     

Identification of consistent novel submegabase deletions in low-grade oligodendrogliomas using array-based comparative genomic hybridization

We have analyzed 18 low-grade gliomas using array comparative genomic hybridization (aCGH) with an average resolution of <500 kb. Because the majority of these tumors showed loss of chromosome arms 1p and 19q, we used custom statistical approaches to define submegabase hemizygous losses throughout the genome that correlated with 19q loss. As a result of this analysis, we have identified a approximately 550-kb region in 11q13 and a approximately 300-kb region in 13q12 that showed hemizygous deletion in virtually all the tumors analyzed regardless of their 1p/19q status. FISH analyses of interphase nuclei from the same tumors used for aCGH analysis confirmed the hemizygous loss. The identification of such specific changes provides a potentially very useful diagnostic marker for this subgroup of low-grade tumors. These regions of the genome define small numbers of candidate genes that are within the deletions. The aCGH analysis also defined the spectrum of gain and loss of genomic regions in low-grade oligodendrogliomas.     

Targeted comparative genomic hybridization array for the detection of single- and multiexon gene deletions and duplications

PurposeTo develop a high resolution microarray based method to detect single- and multiexons gene deletions and duplications.MethodsWe have developed a high-resolution comparative genomic hybridization array to detect single- and multiexon deletions and duplications in a large set of genes on a single microarray, using the NimbleGen 385K array with an exon-centric design.ResultsWe have successfully developed, validated, and implemented a targeted gene comparative genomic hybridization arrays for detecting single- and multiexon deletions and duplication in autosomal and X-linked disease-associated genes.ConclusionThe comparative genomic hybridization arrays can be adopted readily by clinical molecular diagnostic laboratories as a rapid, cost-effective, highly sensitive, and accurate approach for the detection of single- and multiexon deletions or duplications, particularly in cases where direct sequencing fails to identify a mutation.