Microarray-based comparative genomic hybridization and its applications in human genetics

Through the years, several techniques capable of detecting DNA copy number changes have been developed. A number of those, such as karyotyping and fluorescence in situ hybridization (FISH), have proven to be valuable tools in both research and diagnostics. Recently, a new technique, called microarray-based comparative genomic hybridization (array CGH), has been introduced. Array CGH has proven to be a specific, sensitive, and fast technique, with considerable advantages compared to other methods used for the analysis of DNA copy number changes. Array CGH enables analysis of the whole genome in a single experiment. Until now, its applications have been mainly directed at detecting genomic abnormalities in cancer. However, array CGH is also suitable for the analysis of DNA copy number aberrations that cause human genetic disorders. This review gives an overview of array CGH and its applications in human genetics. Advantages, limitations, and future perspectives of array CGH are discussed.     

Fluorescent in situ hybridization and array comparative genomic hybridization: complementary techniques for genomic evaluation

During the past few years a new high-throughput molecular technology, array comparative genomic hybridization, has received a great deal of attention. As a DNA-based tool, this technique is presumably more reproducible than expression arrays. In this review, I discuss how array comparative genomic hybridization is remarkably similar with regard to genome analysis to fluorescent in situ hybridization, a technique that is generally regarded as one of the more accurate and reproducible molecular techniques in diagnostic surgical pathology. A thorough understanding of this technology will be useful for all surgical pathologists in the near future, as this technology will no doubt have some influence on our daily practice.     

Chromosomal imbalances detected by array comparative genomic hybridization in human oligodendrogliomas and mixed oligoastrocytomas

Loss of heterozygosity and fluorescence in situ hybridization (FISH) studies have shown that deletions of 1p and 19q are highly prevalent in oligodendroglioma. However, these tumors have not been comprehensively screened for other alterations in chromosomal dosage. In this study, we used array-based comparative genomic hybridization (CGHa) of mapped BAC DNA to screen for such alterations in 31 oligodendrogliomas (20 grade II, 9 grade III, and 2 grade IV) and 4 mixed oligoastrocytomas (1 grade I, 1 grade II, and 2 grade IV). The most frequent aberrations were loss of 1p (17 cases; 49%) and 19q (15 cases; 43%) and combined loss of 1p/19q (13 cases; 37%). In addition, deletion of 4q, 5p, 9p, 10q, 11p, and 13q was observed in 10, 4, 8, 4, 4, and 13 cases, respectively; loss of whole chromosomes 4, 9, and 13 in 4, 1, and 7 cases, respectively; gain of 7p, 8q, 10p, and 11q in 6, 6, 5, and 10 cases, respectively, and gain of whole chromosomes 7 and 11 in 2 patients each. Minimally altered regions detected by CGHa involved chromosome bands 1p36.32, 4q33, 5p15, 8q24, 11p15, and 19q13.3. Univariate analysis of all 35 cases suggested that combined deletion of 1p and 19q is associated with better survival (P = 0.03). In addition, 8q gain in the oligodendrogliomas was strongly associated with poor outcome (P = 0.002). Also associated with poor disease outcome were alterations that had low prevalence in the pure oligodendrogliomas, including loss of 3q, 9q, and 12q and gain of 1p, 8p, and 10q. In summary, in oligodendrogliomas, CGHa was able to detect novel small alterations in chromosomal dosage that had not been previously detected by other methods. In addition, our findings support the hypotheses that oligodendroglioma can be classified into several groups by CGHa analysis and that specific alterations in genetic dosage may have biologic or clinical significance.     

High-resolution array comparative genomic hybridization analysis of human bronchial and salivary adenoid cystic carcinoma

Adenoid cystic carcinoma (ACC) is a rare but distinctive tumor. Oligonucleotide array comparative genomic hybridization has been applied for cataloging genomic copy number alterations (CNAs) in 17 frozen salivary or bronchial tumors. Only four whole chromosome CNAs were found, and most cases had 2-4 segmental CNAs. No high level amplification was observed. There were recurrent gains at 7p15.2, 17q21-25, and 22q11-13, and recurrent losses at 1p35, 6q22-25, 8q12-13, 9p21, 12q12-13, and 17p11-13. The minimal region of gain at 7p15.2 contained the HOXA cluster. The minimal common regions of deletions contained the CDKN2A/CDKN2B, TP53, and LIMA1 tumor suppressor genes. The recurrent deletion at 8q12.3-13.1 contained no straightforward tumor suppressor gene, but the MIRN124A2 microRNA gene, whose product regulates MMP2 and CDK6. Among unique CNAs, gains harbored CCND1, KIT/PDGFRA/KDR, MDM2, and JAK2. The CNAs involving CCND1, MDM2, KIT, CDKN2A/2B, and TP53 were validated by FISH and/or multiplex ligation-dependent probe amplification. Although most tumors overexpressed cyclin D1 compared with surrounding glands, the only case to overexpress MDM2 had the corresponding CNA. In conclusion, our report suggests that ACC is characterized by a relatively low level of structural complexity. Array CGH and immunohistochemical data implicate MDM2 as the oncogene targeted at 12q15. The gain at 4q12 warrants further exploration as it contains a cluster of receptor kinase genes (KIT/PDGFRA/KDR), whose products can be responsive to specific therapies.     

Large-scale variation among human and great ape genomes determined by array comparative genomic hybridization

Large-scale genomic rearrangements are a major force of evolutionary change and the ascertainment of such events between the human and great ape genomes is fundamental to a complete understanding of the genetic history and evolution of our species. Here, we present the results of an evolutionary analysis utilizing array comparative genomic hybridization (array CGH), measuring copy-number gains and losses among these species. Using an array of 2460 human bacterial artificial chromosomes (BACs) (12% of the genome), we identified a total of 63 sites of putative DNA copy-number variation between humans and the great apes (chimpanzee, bonobo, gorilla, and orangutan). Detailed molecular characterization of a subset of these sites confirmed rearrangements ranging from 40 to at least 175 kb in size. Surprisingly, the majority of variant sites differentiating great ape and human genomes were found within interstitial euchromatin. These data suggest that such large-scale events are not restricted solely to subtelomeric or pericentromeric regions, but also occur within genic regions. In addition, 5/9 of the verified variant sites localized to areas of intrachromosomal segmental duplication within the human genome. On the basis of the frequency of duplication in humans, this represents a 14-fold positional bias. In contrast to previous cytogenetic and comparative mapping studies, these results indicate extensive local repatterning of hominoid chromosomes in euchromatic regions through a duplication-driven mechanism of genome evolution.     

Genomic profiling of myeloid sarcoma by array comparative genomic hybridization

Myeloid sarcoma (MS) is a tumor mass of myeloblasts or immature myeloid cells occurring in an extramedullary site. In this study, seven cases of MS [stomach (1), testis (1), skin (2), and lymph node (3)] and 3 synchronous and 1 follow-up bone marrow (BM) samples were studied for genomic abnormalities using array comparative genomic hybridization (array-CGH). Array-CGH construction used approximately 5,400 bacterial artificial chromosome clones from the RPCI-11 library, spanning the human genome. Data were analyzed using the DNAcopy software and custom heuristics. All MS cases had genomic abnormalities detected by array-CGH. Unbalanced genomic abnormalities in five MS cases were confirmed by conventional cytogenetics (CC) and/or fluorescence in situ hybridization (FISH); these abnormalities included loss of 4q32.1-q35.2, 6q16.1-q21, and 12p12.2-p13.2 and gain of 8q21.2-q24.3, 8, 11q21-q25, 13q21.32-q34, 19, and 21. Array-CGH was also invaluable in identifying possible deletions, partner translocations, and breakpoints that were questionable by CC. The remaining two MS cases had genomic aberrations detected by array-CGH, but were not studied further by CC/FISH. Chromosome 8 was most commonly abnormal (3/7 cases). Identical genomic abnormalities were demonstrated in MS and in synchronous BM in two cases. These results demonstrate that array-CGH is a powerful tool to screen MS tissue for unbalanced genomic abnormalities, allowing identification of chromosome abnormalities when concurrent BM is nonanalyzable or nonleukemic.     

Construction and application of a zebrafish array comparative genomic hybridization platform

The zebrafish is emerging as a prominent model system for studying the genetics of human development and disease. Genetic alterations that underlie each mutant model can exist in the form of single base changes, balanced chromosomal rearrangements, or genetic imbalances. To detect genetic imbalances in an unbiased genome-wide fashion, array comparative genomic hybridization (CGH) can be used. We have developed a 5-Mb resolution array CGH platform specifically for the zebrafish. This platform contains 286 bacterial artificial chromosome (BAC) clones, enriched for orthologous sequences of human oncogenes and tumor suppressor genes. Each BAC clone has been end-sequenced and cytogenetically assigned to a specific location within the zebrafish genome, allowing for ease of integration of array CGH data with the current version of the genome assembly. This platform has been applied to three zebrafish cancer models. Significant genomic imbalances were detected in each model, identifying different regions that may potentially play a role in tumorigenesis. Hence, this platform should be a useful resource for genetic dissection of additional zebrafish developmental and disease models as well as a benchmark for future array CGH platform development.     

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.     

Array-based comparative genomic hybridization analysis of 1176 consecutive clinical genetics investigations

PurposeCytogenetic investigations are useful for etiologic determinations of mental retardation, developmental delay, multiple congenital anomalies, and pregnancy complications; however, the causes remain elusive in a majority of cases despite high-resolution cytogenetic studies and multiple fluorescence in situ hybridization examinations. Array-based comparative genomic hybridization has the ability to examine the genome at a higher resolution and may yield an increased detection of genetic abnormalities. The purpose of this study was to assess the use of array-based comparative genomic hybridization in a clinical genetics setting.MethodsDNA from 1176 patients was analyzed using a bacterial artificial chromosome array-based comparative genomic hybridization platform. All abnormal cases were confirmed by fluorescence in situ hybridization and parental studies were completed when possible.ResultsOf the 1176 patients included in this survey, 163 showed a genomic imbalance identified by array-based comparative genomic hybridization. Of these 163 cases, 116 had a clinically relevant genetic abnormality. A total of 9.8% (116 of 1176 cases) were determined to exhibit a causative genomic imbalance. Twenty-five of the 116 abnormal cases had a previously identified cytogenetic abnormality yielding an increased detection rate of 7.9% (91 of 1146) in cases with normal or no cytogenetics.ConclusionArray-based comparative genomic hybridization increases the overall abnormality detection rate, thus improving the diagnostic potential of clinical cytogenetics investigations.     

Efficient high-resolution deletion discovery in Caenorhabditis elegans by array comparative genomic hybridization

We have developed array Comparative Genomic Hybridization for Caenorhabditis elegans as a means of screening for novel induced deletions in this organism. We designed three microarrays consisting of overlapping 50-mer probes to annotated exons and micro-RNAs, the first with probes to chromosomes X and II, the second with probes to chromosome II alone, and a third to the entire genome. These arrays were used to reliably detect both a large (50 kb) multigene deletion and a small (1 kb) single-gene deletion in homozygous and heterozygous samples. In one case, a deletion breakpoint was resolved to fewer than 50 bp. In an experiment designed to identify new mutations we used the X:II and II arrays to detect deletions associated with lethal mutants on chromosome II. One is an 8-kb deletion targeting the ast-1 gene on chromosome II and another is a 141-bp deletion in the gene C06A8.1. Others span large sections of the chromosome, up to >750 kb. As a further application of array Comparative Genomic Hybridization in C. elegans we used the whole-genome array to detect the extensive natural gene content variation (almost 2%) between the N2 Bristol strain and the strain CB4856, a strain isolated in Hawaii and JU258, a strain isolated in Madeira.     

High-resolution genome-wide array comparative genomic hybridization in splenic marginal zone B-cell lymphoma

Splenic marginal zone B-cell lymphoma is characterized by high genetic heterogeneity, and hepatitis C virus infection seems to be involved in a subset of patients. The aims of the analysis were to identify potential genetic alterations related to hepatitis C virus status, IgV_H gene mutational status, and prognostic categories identified in a multicenter study (Blood 2006;107:4643). Genome-wide array comparative genomic hybridization at a 100-kilobase (kb) resolution was performed in 34 patients with splenic marginal zone B-cell lymphoma, 12 of whom were hepatitis C virus positive. Array-comparative genomic hybridization experiments revealed no copy number alterations in 10 patients (4 were hepatitis C virus positive). A median of 5.6 and 3.8 copy number alterations were detected in hepatitis C virus–positive and in hepatitis C virus–negative patients, respectively. The most frequent copy number alterations involved chromosomes 7 and 17 (21% and 24%, respectively). Except for Xp gain (P = .01), no differences in common alterations were found between hepatitis C virus–positive and hepatitis C virus–negative cases. Unmutated status of the IgV_H gene was related to del(7q) (P = .04) and dup(12q) (P = .03). The high-risk group identified according to the new splenic marginal zone B-cell lymphoma prognostic score was associated with del(7q) (P = .01) and del(17p) (P = .02). Hepatitis C virus–positive splenic marginal zone B-cell lymphoma patients have no specific chromosome alterations. Patients with poor prognosis are characterized by distinctive imbalances.     

Centralization errors in comparative genomic hybridization array analysis of pituitary tumor samples

Reliable interpretation of comparative genomic hybridization array (aCGH) results requires centralization and normalization of the data. We evaluated the reliability of aCGH centralization by comparing aCGH results (with classical centralization‐normalization steps) to fluorescence in situ hybridization (FISH) results. In addition, we propose a method to correct centralization bias. Sixty‐six pituitary tumors were analyzed (Agilent aCGH + SNP 4 × 180K microarray). For each tumor, the FISH‐based log₂(ratios) of a subset of chromosomes were compared with the corresponding aCGH raw log₂(ratios). With our new normalization‐centralization process, this difference was added to all log₂(ratios), before performing loess regression on non‐altered probes only. Finally, the mean log₂(ratio) and the percentage of normal probes were compared between CGHnormaliter and our new FISH‐based method. For 11 tumors, FISH results and raw CGH log₂(ratios) differed significantly. In addition, nine tumors showed discrepancies between results generated by CGHnormaliter and our new‐method. Such discrepancies seemed to occur with tumours with many abnormalities (0%‐40% normal probes), rather than in those tumours with fewer abnormalities (31%‐100% normal probes). Five tumors had too few normal probes to allow normalization. In these tumors, which can exhibit many changes in DNA copy number, we found that centralization bias was frequent and uncorrected by current normalization methods. Therefore, an external control for centralization, such as FISH analysis, is required to insure reliable interpretation of aCGH data.     

Challenges in array comparative genomic hybridization for the analysis of cancer samples

PurposeTo address some of the challenges facing the incorporation of array comparative genomic hybridization technology as a clinical tool, including archived tumor tissue, tumor heterogeneity, DNA quality and quantity, and array comparative genomic hybridization platform selection and performance.MethodsExperiments were designed to assess the impact of DNA source (e.g., archival material), quantity, and amplification on array comparative genomic hybridization results. Two microdissection methods were used to isolate tumor cells to minimize heterogeneity. These data and other data sets were used in a further performance comparison of two commonly used array comparative genomic hybridization platforms: bacterial artificial chromosome (Roswell Park Cancer Institute) and oligonucleotide (Agilent Technologies, Santa Clara, CA).ResultsArray comparative genomic hybridization data from as few as 100 formalin-fixed, paraffin-embedded cells isolated by laser capture microdissection and amplified were remarkably similar to array comparative genomic hybridization copy number alterations detected in the bulk (unamplified) population. Manual microdissection from frozen sections provided a rapid and inexpensive means to isolate tumor from adjacent DNA for amplification and array comparative genomic hybridization. Whole genome amplification introduced no appreciable allele bias on array comparative genomic hybridization. The array comparative genomic hybridization results provided by the bacterial artificial chromosome and Agilent platforms were concordant in general, but bacterial artificial chromosome array comparative genomic hybridization showed far fewer outliers and overall less technical noise, which could adversely affect the statistical interpretation of the data.ConclusionsThis study demonstrates that copy number alterations can be robustly and reproducibly detected by array comparative genomic hybridization in DNA isolated from challenging tumor types and sources, including archival materials, low DNA yield, and heterogeneous tissues. Furthermore, bacterial artificial chromosome array comparative genomic hybridization offers the advantage over the Agilent oligonucleotide platform of presenting fewer outliers, which could affect data interpretation.     

PGD for a complex chromosomal rearrangement by array comparative genomic hybridization

Patients carrying a chromosomal rearrangement (CR) have an increased risk for chromosomally unbalanced conceptions. Preimplantation genetic diagnosis (PGD) may avoid the transfer of embryos carrying unbalanced rearrangements, therefore increasing the chance of pregnancy. Only 7-12 loci can be screened by fluorescence in situ hybridization whereas microarray technology can detect genome-wide imbalances at the single cell level. We performed PGD for a CR carrier with karyotype 46,XY,ins(3;2)(p23;q23q14.2),t(6;14)(p12.2;q13) using array comparative genomic hybridization. Selection of embryos for transfer was only based on copy number status of the chromosomes involved in both rearrangements. In two ICSI-PGD cycles, nine and seven embryos were analysed by array, leaving three and one embryo(s) suitable for transfer, respectively. The sensitivity and specificity of single cell arrays was 100 and 88.8%, respectively. In both cycles a single embryo was transferred, resulting in pregnancy following the second cycle. The embryo giving rise to the pregnancy was normal/balanced for the insertion and translocation but carried a trisomy 8 and nullisomy 9 in one of the two biopsied blastomeres. After 7 weeks of pregnancy the couple miscarried. Genetic analysis following hystero-embryoscopy showed a diploid (90%)/tetraploid (10%) mosaic chorion, while the gestational sac was empty. No chromosome 8 aneuploidy was detected in the chorion, while 8% of the cells carried a monosomy for chromosome 9. In summary, we demonstrate the feasibility and determine the accuracy of single cell array technology to test against transmission of the unbalanced meiotic products that can derive from CRs. Our findings also demonstrate that the genomic constitution of extra-embryonic tissue cannot necessarily be predicted from the copy number status of a single blastomere.     

Recent advances in diagnostic technologies and their impact in autoimmune diseases

Conventional immunological methods for the detection of serum autoantibodies have been an essential tool for the diagnosis of autoimmune diseases for 40 years: in the last decade autoantibody tests have become accepted criteria for the diagnosis and classification of the main systemic and organ-specific autoimmune diseases. The high degree of purification reached by the autoantigens used in these methods has allowed high diagnostic sensitivity and specificity, especially in the case of some new autoantibodies of particular clinical significance, such as anti-nucleosome, anti-transglutaminase, anti-TSH receptor and anti-citrullinated protein autoantibodies. In the last 5 years the advent of proteomic technology, which allows the simultaneous measurement of a number of autoantibodies (multiplexing), has opened up new horizons in the diagnosis of autoimmune diseases. Multiplexing is particularly interesting for clinical laboratories, for organisational, logistical/managerial, physiopathological and research reasons. The emerging technologies are represented by systems based on planar or non-planar (suspension) arrays: the latter include methods which use addressable microbeads or nanobarcoded particles. Within a few years, the new methods will allow testing of individual autoantibody profiles, which will probably improve understanding of the physiopathology of autoimmunity, allow early diagnosis (due to the predictive value of autoantibodies), and drive the diffusion of antigen-specific therapies in autoimmune diseases.     

Recent advances in Bacteroides genetics

Bacteroides are Gram-negative, obligate anaerobes that are present in high concentrations within the intestinal tracts of humans and animals. Bacteroides are also important opportunistic pathogens of humans and animals. Methods for genetic manipulation of these important organisms have only recently begun to emerge. Shuttle vectors which can be transferred by conjugation between Escherichia coli to Bacteroides are now available. A method for transforming some strains of Bacteroides has been developed. Two Bacteroides transposons, Tn4351 and Tn4400, have been found and one of them, Tn4351, has been used for transposon mutagenesis of Bacteroides. Several different Bacteroides genes have now been cloned, including a gene that codes for resistance to clindamycin, genes that code for polysaccharidases (chondroitin lyase and pullulanase), and a gene that codes for a fimbrial subunit. These cloned genes have been used to study the organization and regulation of Bacteroides genes.