Cross-Species Array Comparative Genomic Hybridization Identifies Novel Oncogenic Events in Zebrafish and Human Embryonal Rhabdomyosarcoma

Human cancer genomes are highly complex, making it challenging to identify specific drivers of cancer growth, progression, and tumor maintenance. To bypass this obstacle, we have applied array comparative genomic hybridization (array CGH) to zebrafish embryonal rhabdomyosaroma (ERMS) and utilized cross-species comparison to rapidly identify genomic copy number aberrations and novel candidate oncogenes in human disease. Zebrafish ERMS contain small, focal regions of low-copy amplification. These same regions were commonly amplified in human disease. For example, 16 of 19 chromosomal gains identified in zebrafish ERMS also exhibited focal, low-copy gains in human disease. Genes found in amplified genomic regions were assessed for functional roles in promoting continued tumor growth in human and zebrafish ERMS – identifying critical genes associated with tumor maintenance. Knockdown studies identified important roles for Cyclin D2 (CCND2), Homeobox Protein C6 (HOXC6) and PlexinA1 (PLXNA1) in human ERMS cell proliferation. PLXNA1 knockdown also enhanced differentiation, reduced migration, and altered anchorage-independent growth. By contrast, chemical inhibition of vascular endothelial growth factor (VEGF) signaling reduced angiogenesis and tumor size in ERMS-bearing zebrafish. Importantly, VEGFA expression correlated with poor clinical outcome in patients with ERMS, implicating inhibitors of the VEGF pathway as a promising therapy for improving patient survival. Our results demonstrate the utility of array CGH and cross-species comparisons to identify candidate oncogenes essential for the pathogenesis of human cancer.     

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome ¹. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases ². The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments ². These BAC targets are individually synthesized and spotted in duplicate on a single glass slide ^2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position ^5,6. The SMRT array can detect alterations as small as 50 kb in size ⁷. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection ^7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection ⁸. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.Download video file.^{(62M, mov)}     

DNA Copy Number Aberrations in Breast Cancer by Array Comparative Genomic Hybridization

Array comparative genomic hybridization (CGH) has been popularly used for an-alyzing DNA copy number variations in diseases like cancer. In this study, we investigated 82 sporadic samples from 49 breast cancer patients using 1-Mb reso-lution bacterial artificial chromosome CGH arrays. A number of highly frequent genomic aberrations were discovered, which may act as "drivers" of tumor pro-gression. Meanwhile, the genomic profiles of four "normal" breast tissue samples taken at least 2 cm away from the primary tumor sites were also found to have some genomic aberrations that recurred with high frequency in the primary tu-mors, which may have important implications for clinical therapy. Additionally, we performed class comparison and class prediction for various clinicopathological pa-rameters, and a list of characteristic genomic aberrations associated with different clinicopathological phenotypes was compiled. Our study provides clues for further investigations of the underlying mechanisms of breast carcinogenesis.     

鼻咽癌转移相关基因的比较基因组杂交和cDNA微阵列研究

为筛选在鼻咽癌转移中起重要作用的基因 ,应用比较基因组杂交 (CGH)和cDNA微阵列方法研究成瘤和转移能力不同的鼻咽癌细胞株 6 10B和 5 8F .CGH结果显示 ,2种细胞株在 5p、7q、8p、9q、11p、12q和 17p的一定区域存在差异性扩增 ,在 2q存在差异性缺失 ;cDNA微阵列显示 ,相对于非转移性 6 10B细胞株 ,高转移性 5 8F细胞表现一些基因表达的上调及下调 ,CGH和cDNA微阵列的结合是筛选转移相关基因较好方法     

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient’s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.     

寡核苷酸阵列比较基因组杂交技术及其应用

阵列-比较基因组杂交技术（array comparative genomic hybridization, array CGH）能在全基因组水平和/或高分辨率基础上检测染色体拷贝数的变化,主要应用于遗传学和肿瘤学研究。Array CGH中微阵列探针通常是PCR扩增的BAC克隆或cDNA分子。最近几年,寡核苷酸阵列比较基因组杂交（oligonucleotide array CGH, oaCGH）逐渐开始应用。oaCGH与BAC array CGH比较,具有操作更简便、探针设计更灵活、分辨率更高等多项优点,预计oaCGH将逐步取代利用BAC克隆片段或cDNA分子的array CGH。oaCGH的应用及其与其它高通量检测技术的结合将促进新的癌症相关基因、肿瘤耐药基因的发现。本文综述了现有主要oaCGH平台在空间分辨率、探针长度、灵敏度、特异性等方面的特点及其应用,概括了oaCGH近年来的进展。     

Molecular Karyotyping of Human Single Sperm by Array- Comparative Genomic Hybridization

No valid method is currently available to analyze the entire genome of sperm, including aneuploidies and structural chromosomal alterations. Here we describe the optimization and application of array-Comparative Genomic Hybridization (aCGH) on single human sperm. The aCGH procedure involves screening of the entire chromosome complement by DNA microarray allowing having a molecular karyotype, and it is currently used in research and in diagnostic clinical practice (prenatal diagnosis, pre-implantation genetic diagnosis), but it has never been applied on sperm. DNA from single human sperm isolated by micromanipulator was extracted, decondensed and amplified by whole-genome amplification (WGA) and then labeled, hybridized to BAC array, and scanned by microarray scanner. Application of this protocol to 129 single sperm from normozoospermic donors identified 7.8% of sperm with different genetic anomalies, including aneuploidies and gains and losses in different chromosomes (unbalanced sperm). On the contrary, of 130 single sperm from men affected by Hodgkin lymphoma at the end of three months of chemotherapy cycles 23.8% were unbalanced. Validation of the method also included analysis of 43 sperm from a man with a balanced translocation [46,XY,t(2;12)(p11.2;q24.31)], which showed gains and losses corresponding to the regions involved in the translocation in 18.6% of sperm and alterations in other chromosomes in 16.3% of sperm. Future application of this method might give important information on the biology and pathophysiology of spermatogenesis and sperm chromosome aberrations in normal subjects and in patients at higher risk of producing unbalanced sperm, such as infertile men, carriers of karyotype anomalies, men with advanced age, subjects treated with chemotherapy, and partners of couples with repeated miscarriage and repeated failure during assisted reproduction techniques.     

喉癌的比较基因组杂交研究

寻找与喉癌发生进展的相关基因,应用比较基因组杂交技术分析了18例喉癌患者。结果显示,每例喉癌细胞染色体均有不同程度的变化,包括染色体全部或部分的扩增或丢失。平均每例有12．9处异常区域,丢失多于扩增,分别为每例7．2处和5．7处。主要为3q(78％,)、5p(61％)、11q(56％)、1q(50％,)、8p(44％)、8q(39％)和15q(39％)的扩增;3p(70％)、5q(78％)、90(67%)、13q(50％)、1p(44％)和14q(39％)的丢失。有多条染色体区带上出现特异的扩增或丢失,特别是1p13-21(8／18)、3p21-23(14／18)、5p21-22(14／18)、9p12-pter(10／18)和13q21-31(8／18)的拷贝数增加明显,而1q11-2l(11／18)、3q15-21(12／18)、8p22-24(6／18)、11q12-13(8／18)、15q21-23(7／18)和18p11(8／18)区域的丢失为喉癌的特征性变化,说明这些区域中存在与喉癌发生发展密切相关的癌基因、抑癌基因或其他相关基因。     

Microdeletion and Microduplication Analysis of Chinese Conotruncal Defects Patients with Targeted Array Comparative Genomic Hybridization

Objective

The current study aimed to develop a reliable targeted array comparative genomic hybridization (aCGH) to detect microdeletions and microduplications in congenital conotruncal defects (CTDs), especially on 22q11.2 region, and for some other chromosomal aberrations, such as 5p15-5p, 7q11.23 and 4p16.3.

Methods

Twenty-seven patients with CTDs, including 12 pulmonary atresia (PA), 10 double-outlet right ventricle (DORV), 3 transposition of great arteries (TGA), 1 tetralogy of Fallot (TOF) and one ventricular septal defect (VSD), were enrolled in this study and screened for pathogenic copy number variations (CNVs), using Agilent 8 x 15K targeted aCGH. Real-time quantitative polymerase chain reaction (qPCR) was performed to test the molecular results of targeted aCGH.

Results

Four of 27 patients (14.8%) had 22q11.2 CNVs, 1 microdeletion and 3 microduplications. qPCR test confirmed the microdeletion and microduplication detected by the targeted aCGH.

Conclusion

Chromosomal abnormalities were a well-known cause of multiple congenital anomalies (MCA). This aCGH using arrays with high-density coverage in the targeted regions can detect genomic imbalances including 22q11.2 and other 10 kinds CNVs effectively and quickly. This approach has the potential to be applied to detect aneuploidy and common microdeletion/microduplication syndromes on a single microarray.     

Mutation Screening and Array Comparative Genomic Hybridization Using a 180K Oligonucleotide Array in VACTERL Association

In order to identify genetic causes of VACTERL association (V vertebral defects, A anorectal malformations, C cardiac defects, T tracheoesofageal fistula, E esophageal atresia, R renal anomalies, L limb deformities), we have collected DNA samples from 20 patients diagnosed with VACTERL or with a VACTERL-like phenotype as well as samples from 19 aborted fetal cases with VACTERL. To investigate the importance of gene dose alterations in the genetic etiology of VACTERL association we have performed a systematic analysis of this cohort using a 180K array comparative genomic hybridization (array-CGH) platform. In addition, to further clarify the significance of PCSK5, HOXD13 and CHD7 genes in the VACTERL phenotype, mutation screening has been performed. We identified pathogenic gene dose imbalances in two fetal cases; a hemizygous deletion of the FANCB gene and a (9;18)(p24;q12) unbalanced translocation. In addition, one pathogenic mutation in CHD7 was detected, while no apparent disease-causing mutations were found in HOXD13 or PCSK5. Our study shows that although large gene dose alterations do not seem to be a common cause in VACTERL association, array-CGH is still important in clinical diagnostics to identify disease cause in individual cases.     

Preimplantation Genetic Diagnosis for Aneuploidy and Translocations Using Array Comparative Genomic Hybridization

At least 50% of human embryos are abnormal, and that increases to 80% in women 40 years or older. These abnormalities result in low implantation rates in embryos transferred during in vitro fertilization procedures, from 30% in women <35 years to 6% in women 40 years or older. Thus selecting normal embryos for transfer should improve pregnancy results. The genetic analysis of embryos is called Preimplantation Genetic Diagnosis (PGD) and for chromosome analysis it was first performed using FISH with up to 12 probes analyzed simultaneously on single cells. However, suboptimal utilization of the technique and the complexity of fixing single cells produced conflicting results. PGD has been invigorated by the introduction of microarray testing which allows for the analysis of all 24 chromosome types in one test, without the need of cell fixation, and with staggering redundancy, making the test much more robust and reliable. Recent data published and presented at scientific meetings has been suggestive of increased implantation rates and pregnancy rates following microarray testing, improvements in outcome that have been predicted for quite some time. By using markers that cover most of the genome, not only aneuploidy can be detected in single cells but also translocations. Our validation results indicate that array CGH has a 6Mb resolution in single cells, and thus the majority of translocations can be analyzed since this is also the limit of karyotyping. Even for translocations with smaller exchanged fragments, provided that three out of the four fragments are above 6Mb, the translocation can be detected.     

De Novo Identification of Single Nucleotide Mutations in Caenorhabditis elegans Using Array Comparative Genomic Hybridization

Array comparative genomic hybridization (aCGH) has been used primarily to detect copy-number variants between two genomes. Here we report using aCGH to detect single nucleotide mutations on oligonucleotide microarrays with overlapping 50-mer probes. This technique represents a powerful method for rapidly detecting novel homozygous single nucleotide mutations in any organism with a sequenced reference genome.     

Increasing Live Birth Rate by Preimplantation Genetic Screening of Pooled Polar Bodies Using Array Comparative Genomic Hybridization

Meiotic errors during oocyte maturation are considered the major contributors to embryonic aneuploidy and failures in human IVF treatment. Various technologies have been developed to screen polar bodies, blastomeres and trophectoderm cells for chromosomal aberrations. Array-CGH analysis using bacterial artificial chromosome (BAC) arrays is widely applied for preimplantation genetic diagnosis (PGD) using single cells. Recently, an increase in the pregnancy rate has been demonstrated using array-CGH to evaluate trophectoderm cells. However, in some countries, the analysis of embryonic cells is restricted by law. Therefore, we used BAC array-CGH to assess the impact of polar body analysis on the live birth rate. A disadvantage of polar body aneuploidy screening is the necessity of the analysis of both the first and second polar bodies, resulting in increases in costs for the patient and complex data interpretation. Aneuploidy screening results may sometimes be ambiguous if the first and second polar bodies show reciprocal chromosomal aberrations. To overcome this disadvantage, we tested a strategy involving the pooling of DNA from both polar bodies before DNA amplification. We retrospectively studied 351 patients, of whom 111 underwent polar body array-CGH before embryo transfer. In the group receiving pooled polar body array-CGH (aCGH) analysis, 110 embryos were transferred, and 29 babies were born, corresponding to live birth rates of 26.4% per embryo and 35.7% per patient. In contrast, in the control group, the IVF treatment was performed without preimplantation genetic screening (PGS). For this group, 403 embryos were transferred, and 60 babies were born, resulting in live birth rates of 14.9% per embryo and 22.7% per patient. In conclusion, our data show that in the aCGH group, the use of aneuploidy screening resulted in a significantly higher live birth rate compared with the control group, supporting the benefit of PGS for IVF couples in addition to the suitability and effectiveness of our polar body pooling strategy.     

Genomic Profiling of Oral Squamous Cell Carcinoma by Array-Based Comparative Genomic Hybridization

We designed a study to investigate genetic relationships between primary tumors of oral squamous cell carcinoma (OSCC) and their lymph node metastases, and to identify genomic copy number aberrations (CNAs) related to lymph node metastasis. For this purpose, we collected a total of 42 tumor samples from 25 patients and analyzed their genomic profiles by array-based comparative genomic hybridization. We then compared the genetic profiles of metastatic primary tumors (MPTs) with their paired lymph node metastases (LNMs), and also those of LNMs with non-metastatic primary tumors (NMPTs). Firstly, we found that although there were some distinctive differences in the patterns of genomic profiles between MPTs and their paired LNMs, the paired samples shared similar genomic aberration patterns in each case. Unsupervised hierarchical clustering analysis grouped together 12 of the 15 MPT-LNM pairs. Furthermore, similarity scores between paired samples were significantly higher than those between non-paired samples. These results suggested that MPTs and their paired LNMs are composed predominantly of genetically clonal tumor cells, while minor populations with different CNAs may also exist in metastatic OSCCs. Secondly, to identify CNAs related to lymph node metastasis, we compared CNAs between grouped samples of MPTs and LNMs, but were unable to find any CNAs that were more common in LNMs. Finally, we hypothesized that subpopulations carrying metastasis-related CNAs might be present in both the MPT and LNM. Accordingly, we compared CNAs between NMPTs and LNMs, and found that gains of 7p, 8q and 17q were more common in the latter than in the former, suggesting that these CNAs may be involved in lymph node metastasis of OSCC. In conclusion, our data suggest that in OSCCs showing metastasis, the primary and metastatic tumors share similar genomic profiles, and that cells in the primary tumor may tend to metastasize after acquiring metastasis-associated CNAs.     

Survey of Genomic Diversity among Enterococcus faecalis Strains by Microarray-Based Comparative Genomic Hybridization

We have compared nine Enterococcus faecalis strains with E. faecalis V583 by comparative genomic hybridization using microarrays (CGH). The strains used in this study (the “test” strains) originated from various environments. CGH is a powerful and promising tool for obtaining novel information on genome diversity in bacteria. By CGH, one obtains clues about which genes are present or divergent in the strains, compared to a reference strain (here, V583). The information obtained by CGH is important from both ecological and systematic points of view. CGH of E. faecalis showed considerable diversity in gene content: Compared to V583, the percentage of divergent genes in the test strains varied from 15% to 23%, and 154 genes were divergent in all strains. The main variation was found in regions corresponding to exogenously acquired or mobile DNA in V583. Antibiotic resistance genes, virulence factors, and integrated plasmid genes dominated among the divergent genes. The strains examined showed various contents of genes corresponding to the pTEF1, pTEF2, and pTEF3 genes in V583. The extensive transport and metabolic capabilities of V583 appeared similar in the test strains; CGH indicated that the ability to transport and metabolize various carbohydrates was similar in the test strains (verified by API 50 CH assays). The contents of genes related to stress tolerance appeared similar in V583 and the nine test strains, supporting the view of E. faecalis as an organism able to resist harsh conditions.     

Diversity Suppression-Subtractive Hybridization Array for Profiling Genomic DNA Polymorphisms

Genomlc DNA polymorphlsms are very useful for tracing genetic traits end studying biological diversity among species. Here, we present a method we call the ＂diversity suppresslon-subtractlve hybridization array＂ for effectively profiling genomlc DNA polymorphisms. The method first obtains the subtracted gDNA fragments between any two species by suppression subtraction hybridization （SSH） to establish e subtracted gDNA library, from which diversity SSH arrays are created with the selected subtracted clones. The diversity SSH array hybridizes with the DIG-labeled genomlc DNA of the organism to be assayed. Six closely related Dendrobium species were studied as model samples. Four Dendrobium species as testers were used to perform SSH. A total of 617 subtracted positive clones were obtained from four Dendrobium species, and the average ratio of positive clones was 80.3%. We demonstrated that the average percentage of polymorphlc fragments of palrwlse comparisons of four Dendrobium species was up to 42.4%. A dendrogram of the relatedness of six Dendrobium species was produced according to their polymorphic profiles. The results revealed that the diversity SSH array Is a highly effective platform for profiling genomlc DNA polymorphlsms and dendrograms.     

Identification of Genomic Alterations in Pancreatic Cancer Using Array-Based Comparative Genomic Hybridization

Background

Genomic aberration is a common feature of human cancers and also is one of the basic mechanisms that lead to overexpression of oncogenes and underexpression of tumor suppressor genes. Our study aims to identify frequent genomic changes in pancreatic cancer.

Materials and Methods

We used array comparative genomic hybridization (array CGH) to identify recurrent genomic alterations and validated the protein expression of selected genes by immunohistochemistry.

Results

Sixteen gains and thirty-two losses occurred in more than 30% and 60% of the tumors, respectively. High-level amplifications at 7q21.3–q22.1 and 19q13.2 and homozygous deletions at 1p33–p32.3, 1p22.1, 1q22, 3q27.2, 6p22.3, 6p21.31, 12q13.2, 17p13.2, 17q21.31 and 22q13.1 were identified. Especially, amplification of AKT2 was detected in two carcinomas and homozygous deletion of CDKN2C in other two cases. In 15 independent validation samples, we found that AKT2 (19q13.2) and MCM7 (7q22.1) were amplified in 6 and 9 cases, and CAMTA2 (17p13.2) and PFN1 (17p13.2) were homozygously deleted in 3 and 1 cases. AKT2 and MCM7 were overexpressed, and CAMTA2 and PFN1 were underexpressed in pancreatic cancer tissues than in morphologically normal operative margin tissues. Both GISTIC and Genomic Workbench software identified 22q13.1 containing APOBEC3A and APOBEC3B as the only homozygous deletion region. And the expression levels of APOBEC3A and APOBEC3B were significantly lower in tumor tissues than in morphologically normal operative margin tissues. Further validation showed that overexpression of PSCA was significantly associated with lymph node metastasis, and overexpression of HMGA2 was significantly associated with invasive depth of pancreatic cancer.

Conclusion

These recurrent genomic changes may be useful for revealing the mechanism of pancreatic carcinogenesis and providing candidate biomarkers.     

Spectrum of Cytogenomic Abnormalities Revealed by Array Comparative Genomic Hybridization on Products of Conception Culture Failure and Normal Karyotype Samples

Approximately 30% of pregnancies after implantation end up in spontaneous abortions, and 50% of them are caused by chromosomal abnormalities. However, the spectrum of genomic copy number variants(CNVs) in products of conception(POC) and the underlying genedosage-sensitive mechanisms causing spontaneous abortions remain largely unknown. In this study, array comparative genomic hybridization(a CGH) analysis was performed as a salvage procedure for 128 POC culture failure(POC-CF) samples and as a supplemental procedure for106 POC normal karyotype(POC-NK) samples. Chromosomal abnormalities were detected in 10% of POC-CF and pathogenic CNVs were detected in 3.9% of POC-CF and 5.7% of POC-NK samples. Compiled results from this study and relevant case series through a literature review demonstrated an abnormality detection rate(ADR) of 35% for chromosomal abnormalities in POC-CF samples, 3.7% for pathogenic CNVs in POC-CF samples, and 4.6% for pathogenic CNVs in POC-NK samples. Ingenuity Pathway Analysis(IPA) was performed on the genes from pathogenic CNVs found in POC samples. The denoted primary gene networks suggested that apoptosis and cell proliferation pathways are involved in miscarriage. In summary, a similar spectrum of cytogenomic abnormalities was observed in POC culture success and POC-CF samples. A threshold effect correlating the number of dosage-sensitive genes in a chromosome with the observed frequency of autosomal trisomy is proposed. A rationalized approach using firstly fluorescence in situ hybridization(FISH) testing with probes of chromosomes X/Y/18, 13/21, and 15/16/22 for common aneuploidies and polyploidies and secondly a CGH for other cytogenomic abnormalities is recommended for POC-CF samples.     

西瓜与近缘种亲缘关系的比较基因组原位杂交分析

物种间亲缘关系的研究是杂交育种的理论基础,野生西瓜在西瓜育种中具有重要作用,然而目前对西瓜属物种间亲缘关系的研究十分有限,而且对西瓜属物种的分类问题还存在分歧.比较基因组原位杂交是分析物种间亲缘关系的有效手段,本研究以西瓜基因组DNA作探针,分别对缺须西瓜、热迷西瓜、药西瓜和诺丹西瓜有丝分裂中期染色体进行了比较基因组原位杂交分析,揭示了西瓜属物种间的亲缘关系,同时对分类地位尚存在争议的诺丹西瓜的归属问题进行了分析,发现诺丹西瓜和甜瓜之间具有非常近的亲缘关系,本研究结果为西瓜与近缘种间的远缘杂交提供了重要的理论依据.