Cytogenetics in reproductive medicine: the contribution of comparative genomic hybridization (CGH)

Cytogenetic research has had a major impact on the field of reproductive medicine, providing an insight into the frequency of chromosomal abnormalities that occur during gametogenesis, embryonic development and pregnancy. In humans, aneuploidy has been found to be relatively common during fetal life, necessitating prenatal screening of high-risk pregnancies. Aneuploidy rates are higher still during the preimplantation stage of development. An increasing number of IVF laboratories have attempted to improve pregnancy rates by using preimplantation genetic diagnosis (PGD) to ensure that the embryos transferred to the mother are chromosomally normal. This paper reviews some of the techniques that are key to the detection of aneuploidy in reproductive samples including comparative genomic hybridization (CGH). CGH has provided an unparalleled insight into the nature of chromosome imbalance in human embryos and polar bodies. The clinical application of CGH for the purposes of PGD and the future extensions of the methodology, including DNA microarrays, are discussed.     

Moth sex chromatin probed by comparative genomic hybridization (CGH).

Abstract: Comparative genomic hybridization (CGH) with a probe mixture of differently labeled genomic DNA from females and males highlighted the W chromosomes in mitotic plates and the W chromatin in polyploid interphase nuclei of the silkworm Bombyx mori, the flour moth Ephestia kuehniella, and the wax moth Galleria mellonella. The overproportionate fluorescence signal indicated an accumulation of repetitive sequences in the respective W chromosomes. Measurements of the fluorescence signals revealed two components, one that is present also in male DNA (non-W chromosomes) and another one that is present only in or preponderantly in female DNA (W chromosomes). While the W chromosomes of E. kuehniella and G. mellonella had both components, that of B. mori appeared to lack the latter component. Our results show that CGH can be applied to obtain a first estimate of the sequence composition of sex chromosomes in species from which otherwise little is known on the molecular level.     

High-resolution molecular characterization of 15q11-q13 rearrangements by array comparative genomic hybridization (array CGH) with detection of gene dosage

Maternally derived duplication of the imprinted region of chromosome 15q11-q14 leads to a complex neurobehavioral phenotype that often includes autism, cognitive deficits, and seizures. Multiple repeat elements within the region mediate a variety of rearrangements, including interstitial duplications, interstitial triplications, and supernumerary isodicentric marker chromosomes, as well as the deletions that cause Prader-Willi and Angelman syndromes. To elucidate the molecular structure of these duplication chromosomes, we designed a high-resolution array comparative genomic hybridization (array CGH) platform. The array contains 79 clones that form a gapped contig across the critical region on chromosome 15q11-q14 and 21 control clones from other autosomes and the sex chromosomes. We used this array to examine a set of 48 samples from patients with segmental aneuploidy of chromosome 15q. Using the array, we were able to determine accurately the dosage, which ranged from 1 to 6 copies, and also to detect atypical and asymmetric rearrangements. In addition, the increased resolution of the array allowed us to position two previously reported breakpoints within the contig. These results indicate that array CGH is a powerful technique to study rearrangements of proximal chromosome 15q.     

Cluster analysis of comparative genomic hybridization (CGH) data using self-organizing maps: application to prostate carcinomas.

Comparative genomic hybridization (CGH) is a modern genetic method which enables a genome-wide survey of chromosomal imbalances. For each chromosome region, one obtains the information whether there is a loss or gain of genetic material, or whether there is no change at that region. Usually it is not possible to evaluate all 46 chromosomes of a metaphase, therefore several (up to 20 or more) metaphases are analyzed per individual, and expressed as average. Mostly one does not study one individual alone but groups of 20-30 individuals. Therefore, large amounts of data quickly accumulate which must be put into a logical order. In this paper we present the application of a self-organizing map (Genecluster) as a tool for cluster analysis of data from pT2N0 prostate cancer cases studied by CGH. Self-organizing maps are artificial neural networks with the capability to form clusters on the basis of an unsupervised learning rule, i.e., in our examples it gets the CGH data as only information (no clinical data). We studied a group of 40 recent cases without follow-up, an older group of 20 cases with follow-up, and the data set obtained by pooling both groups. In all groups good clusterings were found in the sense that clinically similar cases were placed into the same clusters on the basis of the genetic information only. The data indicate that losses on chromosome arms 6q, 8p and 13q are all frequent in pT2N0 prostatic cancer, but the loss on 8p has probably the largest prognostic importance.     

Genome-wide screening of severe male factor infertile patients using BAC-array comparative genomic hybridization (CGH)

Male factor infertility is present in up to 50% of infertile couples, making it increasingly important in their treatment. Although most research into the genetics of male infertility has focused on the Y chromosome, male factor infertility may result from other genetic factors. We utilized the whole genome array comparative genomic hybridization (CGH) to identify novel genetic candidate associated with severely impaired spermatogenesis. We enrolled 37 patients with severe male factor infertility, defined as severe nonobstructive type oligozoospermia (≤5×10(6)/ml) or azoospermia, and 10 controls. Routine cytogenetic analyses, Yq microdeletion PCR test and whole genome bacterial artificial chromosome (BAC)-array CGH were performed. Array CGH results showed no specific gains or losses related to impaired spermatogenesis other than Yq microdeletions, and there were no novel candidate genetic abnormalities in the patients with severe male infertility. However, Yq microdeletions were detected in 10 patients. Three showed a deletion in the AZFb-c region and the other 7 had deletions in the AZFc region. Although we could not identify novel genetic regions specifically associated with male infertility, whole genome array CGH analysis with higher resolution including larger numbers of patients may be able to give an opportunity for identifying new genetic markers for male infertility.     

DNA profiling by array comparative genomic hybridization (CGH) of peripheral blood mononuclear cells (PBMC) and tumor tissue cell in non-small cell lung cancer (NSCLC)

Lung tumor cell DNA copy number alteration (CNA) was expected to display specific patterns such as a large-scale amplification or deletion of chromosomal arms, as previously published data have reported. Peripheral blood mononuclear cell (PBMC) CNA however, was expected to show normal variations in cancer patients as well as healthy individuals, and has thus been used as normal control DNA samples in various published studies. We performed array CGH to measure and compare genetic changes in terms of the CNA of PBMC samples as well as DNA isolated from tumor tissue samples, obtained from 24 non-small cell lung cancer patients. Contradictory to expectations, our studies showed that the PBMC CNA also showed chromosomal variant regions. The list included well-known tumor-associated NTRK1, FGF8, TP53, and TGFβ1 genes and potentially novel oncogenes such as THPO (3q27.1), JMJD1B, and EGR1 (5q31.2), which was investigated in this study. The results of this study highlighted the connection between PBMC and tumor cell genomic DNA in lung cancer patients. However, the application of these studies to cancer prognosis may pose a challenge due to the large amount of information contained in genetic predisposition and family history that has to be processed for useful downstream clinical applications.     

A simple method for enhancing hybridization efficiency in chromosome and array comparative genomic hybridization

Abstract

The accuracy of comparative genomic hybridization (CGH) analysis is affected by hybridization efficiency. We describe here a simple method for enhancing hybridization efficiency. The hybridization procedure is essentially the same as that of conventional methods. Hybridization solution containing denatured DNA probe mixture was applied to a metaphase chromosome slide or DNA chip slide and covered with a coverslip. In the new method, however, the slide was inverted by turning the coverslip downward prior to hybridization. We termed this method the inverted slide method. To estimate the efficiency of the new method, metaphase chromosome slides and DNA chip slides were treated by both the conventional and inverted slide methods and incubated in a moist chamber at 37°C for 12, 24, 48, and 72 h. Hybridization signals were approximately 1.5 to 2 times brighter on the slides using the inverted slide method than those using the conventional method after 48 and 72 h of incubation. Furthermore, topographical differences in fluorescence intensity were smaller in slides using the inverted-slide method than in those prepared by the conventional method. The inverted slide method is methodologically very simple and improves the resolution of CGH.     

From array to array: Confirmation of genomic gains and losses discovered by array-based comparative genomic hybridization utilizing fluorescence in situ hybridization on tissue microarrays

The combination of array-based comparative genomic hybridization (CGH) with fluorescence in situ hybridization utilizing custom-designed bacterial artificial chromosome (BAC) probes applied to tissue microarrays represents a powerful compendium of techniques–greatly enhancing the throughput of genomic analysis and subsequent target validation. Such approach can be automated at various levels and allows managing large volume of targets and samples in a few experiments. As such, this approach facilitates discovery, validation and implementation of findings in the process of identification of new diagnostic, prognostic and potentially therapeutic molecular markers.     

Application of array comparative genomic hybridization in 256 patients with developmental delay or intellectual disability

We used whole-genome exon-targeted oligonucleotide array comparative genomic hybridization (array CGH) in a cohort of 256 patients with developmental delay (DD)/intellectual disability (ID) with or without dysmorphic features, additional neurodevelopmental abnormalities, and/or congenital malformations. In 69 patients, we identified 84 non-polymorphic copy-number variants, among which 41 are known to be clinically relevant, including two recently described deletions, 4q21.21q21.22 and 17q24.2. Chromosomal microarray analysis revealed also 15 potentially pathogenic changes, including three rare deletions, 5q35.3, 10q21.3, and 13q12.11. Additionally, we found 28 copy-number variants of unknown clinical significance. Our results further support the notion that copy-number variants significantly contribute to the genetic etiology of DD/ID and emphasize the efficacy of the detection of novel candidate genes for neurodevelopmental disorders by whole-genome array CGH.     

CGcgh: a tool for molecular karyotyping using DNA microarray-based comparative genomic hybridization (array-CGH)

Microarray-based comparative genomic hybridization (array-CGH) is a technique by which variations in copy numbers between two genomes can be analyzed using DNA microarrays. Array CGH has been used to survey chromosomal amplifications and deletions in fetal aneuploidies or cancer tissues. Herein we report a user-friendly, MATLAB-based, array CGH analyzing program, Chang Gung comparative genomic hybridization (CGcgh), as a standalone PC version. The analyzed chromosomal data are displayed in a graphic interface, and CGcgh allows users to launch a corresponding G-banding ideogram. The abnormal DNA copy numbers (gains and losses) can be identified automatically using a user defined window size (default value is 50 probes) and sequential student t-tests with sliding windows along with chromosomes. CGcgh has been tested in multiple karyotype-confirmed human samples, including five published cases and trisomies 13, 18, 21 and X from our laboratories, and 18 cases of which microarray data are available publicly. CGcgh can be used to detect the copy number changes in small genomic regions, which are commonly encountered by clinical geneticists. CGcgh works well for the data from cDNA microarray, spotted oligonucleotide microarrays, and Affymetrix Human Mapping Arrays (10K, 100K, 500K Array Sets). The program can be freely downloaded from . Y. S. Lee and A. Chao contributed equally to this work.     

Genomic profiling of plasmablastic lymphoma using array comparative genomic hybridization (aCGH): revealing significant overlapping genomic lesions with diffuse large B-cell lymphoma

Background

Cholestatic jaundice as a presenting symptom of Precursor T-lymphoblastic leukemia (T-ALL)/lymphoma (T-LBL) has never been reported in literature. Similarly, precursor T-ALL/T-LBL is characteristically negative for synaptophysin. We report the first case of a patient with precursor T-ALL/T-LBL who presented with cholestatic jaundice and aberrant tumor expression of synaptophysin.

Case report

42 year old male presented with anorexia, nausea, jaundice, pale stools, dark urine and about 35 pound weight loss over the previous 3 weeks. The initial laboratory work was suggestive of cholestatic jaundice. Markedly elevated LDH (2025 U/L) and CA 19-9 (1778 u/ML) were also noticed. The CT scan of abdomen showed massive hepatomegaly with coarse echotexture with contracted gall bladder and normal sized common bile duct. Chest x-ray revealed a mediastinal mass with mediastinal widening. CT scan of the chest showed anterior mediastinal mass (16 cm × 10 cm). CT guided biopsy of the mass showed malignant lymphoma with diffuse proliferation of medium sized lymphoid cells. The neoplastic cells were positive for CD1a, CD3, CD4, CD5, CD8 and CD43 with aberrant expression of synaptophysin. PET CT scan again showed a large anterior mediastinal mass with diffuse liver involvement and abnormal activity in axial bones. CT guided liver biopsy and bone marrow biopsy revealed the same morphology and immunohistochemistry. Bone marrow aspirate showed 85% lymphoblasts. Thus, the diagnosis of precursor T-ALL/T-LBL was made and jaundice with elevated CA 19-9 were attributed to intrahepatic cholestasis.

Conclusion

Our case illustrates an unusual presentation of hematological malignancies as cholestatic jaundice. It also indicates the non-specific nature of CA 19-9 for pancreaticobiliary malignancies. It is the first case report of neoplastic precursor T cell lymphoblasts with unusual expression of synaptophysin. Tissue biopsy with thorough immunohistochemistry is required to differentiate precursor T-ALL/T-LBL from thymoma and small cell carcinoma.     

Comparative genomic hybridization (CGH): ten years of substantial progress in human solid tumor molecular cytogenetics

Data from ten years of research using comparative genomic hybridization (CGH) for the detection of chromosomal alterations in human solid tumors are concisely reviewed. By use of a basic methodology with some variations more or less specific patterns of genomic imbalances were found in a large number of tumors of various entities. Specific gains and losses of genomic material have not only opened the way to the detection of a series of cancer-related genes but also to clinical implications. Not only several areas of basic oncogenetic research, but also differential diagnosis, prognosis of disease progression, and therapeutic decisions have profited by CGH.     

SW-ARRAY: a dynamic programming solution for the identification of copy-number changes in genomic DNA using array comparative genome hybridization data

Comparative genome hybridization (CGH) to DNA microarrays (array CGH) is a technique capable of detecting deletions and duplications in genomes at high resolution. However, array CGH studies of the human genome noting false negative and false positive results using large insert clones as probes have raised important concerns regarding the suitability of this approach for clinical diagnostic applications. Here, we adapt the Smith–Waterman dynamic-programming algorithm to provide a sensitive and robust analytic approach (SW-ARRAY) for detecting copy-number changes in array CGH data. In a blind series of hybridizations to arrays consisting of the entire tiling path for the terminal 2 Mb of human chromosome 16p, the method identified all monosomies between 267 and 1567 kb with a high degree of statistical significance and accurately located the boundaries of deletions in the range 267–1052 kb. The approach is unique in offering both a nonparametric segmentation procedure and a nonparametric test of significance. It is scalable and well-suited to high resolution whole genome array CGH studies that use array probes derived from large insert clones as well as PCR products and oligonucleotides.     

A cytogenetically characterized, genome-anchored 10-Mb BAC set and CGH array for the domestic dog

The generation of a 7.5x dog genome assembly provides exciting new opportunities to interpret tumor-associated chromosome aberrations at the biological level. We present a genomic microarray for array comparative genomic hybridization (aCGH) analysis in the dog, comprising 275 bacterial artificial chromosome (BAC) clones spaced at intervals of approximately 10 Mb. Each clone has been positioned accurately within the genome assembly and assigned to a unique chromosome location by fluorescence in situ hybridization (FISH) analysis, both individually and as chromosome-specific BAC pools. The microarray also contains clones representing the dog orthologues of 31 genes implicated in human cancers. FISH analysis of the 10-Mb BAC clone set indicated excellent coverage of each dog chromosome by the genome assembly. The order of clones was consistent with the assembly, but the cytogenetic intervals between clones were variable. We demonstrate the application of the BAC array for aCGH analysis to identify both whole and partial chromosome imbalances using a canine histiocytic sarcoma case. Using BAC clones selected from the array as probes, multicolor FISH analysis was used to further characterize these imbalances, revealing numerous structural chromosome rearrangements. We outline the value of a combined aCGH/FISH approach, together with a well-annotated dog genome assembly, in canine and comparative cancer studies.     

Chromosomal 16p microdeletion in Rubinstein-Taybi syndrome detected by oligonucleotide-based array comparative genomic hybridization: a case report

Introduction

Chromosomal aberrations of chromosome 16 are uncommon and submicroscopic deletions have rarely been reported. At present, a cytogenetic or molecular abnormality can only be detected in 55% of Rubinstein-Taybi syndrome patients, leaving the diagnosis in 45% of patients to rest on clinical features only. Interestingly, this microdeletion of 16 p13.3 was found in a young child with an unexplained syndromic condition due to an indistinct etiological diagnosis. To the best of our knowledge, no evidence of a microdeletion of 16 p13.3 with contiguous gene deletion, comprising cyclic adenosine monophosphate-response element-binding protein and tumor necrosis factor receptor-associated protein 1 genes, has been described in typical Rubinstein-Taybi syndrome.

Case presentation

We present the case of a three-year-old Malaysian Chinese girl with a de novo microdeletion on the short arm of chromosome 16, identified by oligonucleotide array-based comparative genomic hybridization. Our patient showed mild to moderate global developmental delay, facial dysmorphism, bilateral broad thumbs and great toes, a moderate size atrial septal defect, hypotonia and feeding difficulties. A routine chromosome analysis on 20 metaphase cells showed a normal 46, XX karyotype. Further investigation by high resolution array-based comparative genomic hybridization revealed a 120 kb microdeletion on chromosomal band 16 p13.3.

Conclusion

A mutation or abnormality in the cyclic adenosine monophosphate-response element-binding protein has previously been determined as a cause of Rubinstein-Taybi syndrome. However, microdeletion of 16 p13.3 comprising cyclic adenosine monophosphate-response element-binding protein and tumor necrosis factor receptor-associated protein 1 genes is a rare scenario in the pathogenesis of Rubinstein-Taybi syndrome. Additionally, due to insufficient coverage of the human genome by conventional techniques, clinically significant genomic imbalances may be undetected in unexplained syndromic conditions of young children. This case report demonstrates the ability of array-based comparative genomic hybridization to offer a genome-wide analysis at high resolution and provide information directly linked to the physical and genetic maps of the human genome. This will contribute to more accurate genetic counseling and provide further insight into the syndrome.     

Gene copy number changes in dermatofibrosarcoma protuberans - a fine-resolution study using array comparative genomic hybridization

Dermatofibrosarcoma protuberans (DFSP) is a rare, slow-growing, low-grade dermal tumor. Cytogenetic and FISH studies have revealed that the chromosomal rearrangements characteristic of DFSP tumors involve both translocations and the formation of a supernumerary ring derived from chromosomes 17 and 22. The t(17;22) (q22;q13.1) translocation generates a gene fusion between COL1A1 and PDGFB, which serves as a diagnostic marker of DFSP. In the present study we performed array-CGH (aCGH) analysis on ten DFSP tumors. The COL1A1 region at 17q was gained in 71% (5/7) of the samples and the PDGFB region at 22q was gained in 43% (3/7) of the individual samples. In addition to the 17q and 22q gains, altogether 17 minimal common regions of gain and one region of loss were detected.     

CGHMultiArray: exact P-values for multi-array comparative genomic hybridization data

We compute P-values, based on the Wilcoxon test with ties, to compare two conditions with array comparative genomic hybridization data, and we provide a simple interface to export and plot these P-values.     

GeneCount: genome-wide calculation of absolute tumor DNA copy numbers from array comparative genomic hybridization data

Absolute tumor DNA copy numbers can currently be achieved only on a single gene basis by using fluorescence in situ hybridization (FISH). We present GeneCount, a method for genome-wide calculation of absolute copy numbers from clinical array comparative genomic hybridization data. The tumor cell fraction is reliably estimated in the model. Data consistent with FISH results are achieved. We demonstrate significant improvements over existing methods for exploring gene dosages and intratumor copy number heterogeneity in cancers.     

DNA ploidy and chromosomal imbalances in invasive ductal breast cancer. A comparative study of DNA image cytometry and comparative genomic hybridization (CGH).

Chromosomal imbalances were analyzed in 62 breast cancers with different DNA ploidy by CGH. The results of DNA image cytometry and CGH are consistent with peridiploid and aneuploid cases. The peritetraploid tumors harbored a high number of chromosomal imbalances, as a hint for an unfavorable prognosis. The quantitative analysis of imbalances highlighted the role of different physical constituents of the chromosome, and of chromosomal losses in different DNA ploidy groups. The peritetraploid and aneuploid tumors differed from the peridiploid tumors in losses at 8p and 18q. The peritetraploid cancers exhibited more gains at 8q, the aneuploid tumors more losses at 17p than their peridiploid counterparts. The aneuploid cases differed from the peritetraploid tumors in a higher number of losses at 11q and 14q. Combinations of imbalances provide further insights into the genetic background of DNA ploidy. Hypotheses for the progression from peridiploid to nondiploid breast cancers are given.     

Development of a DNA-labeling system for array-based comparative genomic hybridization.

Chromosomal amplifications and deletions are critical components of tumorigenesis and DNA copy-number variations also correlate with changes in mRNA expression levels. Genome-wide microarray comparative genomic hybridization (CGH) has become an important method for detecting and mapping chromosomal changes in tumors. Thus, the ability to detect twofold differences in fluorescent intensity between samples on microarrays depends on the generation of high-quality labeled probes. To enhance array-based CGH analysis, a random prime genomic DNA labeling method optimized for improved sensitivity, signal-to-noise ratios, and reproducibility has been developed. The labeling system comprises formulated random primers, nucleotide mixtures, and notably a high concentration of the double mutant exo-large fragment of DNA polymerase I (exo-Klenow). Microarray analyses indicate that the genomic DNA-labeled templates yield hybridization signals with higher fluorescent intensities and greater signal-to-noise ratios and detect more positive features than the standard random prime and conventional nick translation methods. Also, templates generated by this system have detected twofold differences in gene copy number between male and female genomic DNA and identified amplification and deletions from the BT474 breast cancer cell line in microarray hybridizations. Moreover, alterations in gene copy number were routinely detected with 0.5 microg of genomic DNA starting sample. The method is flexible and performs efficiently with different fluorescently labeled nucleotides. Application of the optimized CGH labeling system may enhance the resolution and sensitivity of array-based CGH analysis in cancer and medical genetic studies.