Novel applications of array comparative genomic hybridization in molecular diagnostics.

INTRODUCTION: In 2004, the implementation of array comparative genomic hybridization (array comparative genome hybridization [CGH]) into clinical practice marked a new milestone for genetic diagnosis. Array CGH and single-nucleotide polymorphism (SNP) arrays enable genome-wide detection of copy number changes in a high resolution, and therefore microarray has been recognized as the first-tier test for patients with intellectual disability or multiple congenital anomalies, and has also been applied prenatally for detection of clinically relevant copy number variations in the fetus. Area covered: In this review, the authors summarize the evolution of array CGH technology from their diagnostic laboratory, highlighting exonic SNP arrays developed in the past decade which detect small intragenic copy number changes as well as large DNA segments for the region of heterozygosity. The applications of array CGH to human diseases with different modes of inheritance with the emphasis on autosomal recessive disorders are discussed. Expert commentary: An exonic array is a powerful and most efficient clinical tool in detecting genome wide small copy number variants in both dominant and recessive disorders. However, whole-genome sequencing may become the single integrated platform for detection of copy number changes, single-nucleotide changes as well as balanced chromosomal rearrangements in the near future.

aCGH-MAS: analysis of aCGH by means of multiagent system.

There are currently different techniques, such as CGH arrays, to study genetic variations in patients. CGH arrays analyze gains and losses in different regions in the chromosome. Regions with gains or losses in pathologies are important for selecting relevant genes or CNVs (copy-number variations) associated with the variations detected within chromosomes. Information corresponding to mutations, genes, proteins, variations, CNVs, and diseases can be found in different databases and it would be of interest to incorporate information of different sources to extract relevant information. This work proposes a multiagent system to manage the information of aCGH arrays, with the aim of providing an intuitive and extensible system to analyze and interpret the results. The agent roles integrate statistical techniques to select relevant variations and visualization techniques for the interpretation of the final results and to extract relevant information from different sources of information by applying a CBR system.

[Familial presentation of microdeletion and inverted microduplication with array-CGH]. / Microdelecion y microduplicacion inversa de presentacion familiar con array-CGH.

INTRODUCTION. Over the years the field of genetics has advanced significantly. Following the polymerase chain reaction and mass sequencing techniques, the array-CGH technique (comparative genomic hybridization) has helped to improve genetic procedures. A resolution of up to 200 kb is currently being accomplished in the human genome. CASE REPORTS. We report the case of two sisters with delays in developmental milestones and a characteristic phenotype with normal results from initial studies of the karyotype and subtelomeric regions. Array-CGH was later used to detect a deletion and duplication that were different in each of the sisters, this being the result of a balanced paternal translocation. In the two cases, despite being the result of the same translocation, the genetic and phenotype expression were different. CONCLUSIONS. The precision achieved by means of array-CGH is making it possible to establish a correlation between minimum gains or losses of the genome and the clinical features. Chromosome 3 codes for genes that play a fundamental role in neurological development (contactins, neurotransmitter modulator proteins, etc.) and chromosome 10 codes for proteins involved in apoptosis and proteins regulating transcription. In the literature there have been reports of chromosome 3 deletion syndrome and monosomy 10. Likewise, there are also descriptions of rearrangements between these chromosomes in individuals from the same family. Nevertheless, we describe two cases of a family with a micro-deletion and an inverted microduplication, detected by means of array-CGH, that have not been reported to date. This technique can provide a diagnostic and prognostic approximation as regards development and offer genetic counselling.

TITLE: Microdelecion y microduplicacion inversa de presentacion familiar con array-CGH.Introduccion. A lo largo de los años se han logrado avances en torno a la genetica; tras la reaccion en cadena de la polimerasa y las tecnicas de secuenciacion masiva, la tecnica array-CGH (comparative genomic hybridization) ha contribuido a mejorar los procedimientos geneticos. Actualmente esta consiguiendo una resolucion de hasta 200 kb en el genoma humano. Casos clinicos. Se presentan dos hermanas con retraso en los hitos del desarrollo y fenotipo caracteristico con estudio inicial de cariotipo y de regiones subtelomericas normales. Posteriormente, mediante array-CGH se detecto en cada una una delecion y una duplicacion diferentes, fruto de una translocacion equilibrada paterna. En ambas, siendo fruto de una misma translocacion, muestra diferente expresion genetica y fenotipica. Conclusiones. La precision conseguida mediante el array-CGH esta permitiendo correlacionar minimas ganancias o perdidas del genoma con la clinica. En el cromosoma 3 se encuentran codificados genes fundamentales en el desarrollo neurologico (contactinas, proteinas moduladoras de neurotransmisores…), y en el cromosoma 10, proteinas implicadas en la apoptosis y proteinas reguladoras de la transcripcion. En la bibliografia se han descrito el sindrome de delecion del cromosoma 3 y la monosomia 10. Igualmente, hay descritos reordenamientos entre estos cromosomas en individuos de una misma familia. Sin embargo, aportamos dos casos de una familia con una microdelecion y una microduplicacion inversa, detectados mediante array-CGH, no descritos hasta el momento. Dicha tecnica puede ofrecer una aproximacion diagnostica y pronostica en cuanto a la evolucion y ofertar consejo genetico.

Analysis of Copy-Number Alterations in Single Cells Using Microarray-Based Comparative Genomic Hybridization (aCGH).

In this unit, we describe a workflow that enables array comparative genomic hybridization (aCGH) of single cells. The unit first describes the isolation and preparation of single peripheral mononuclear cells from blood (PBMC) to prepare a suitable reference DNA for aCGH experiments. An alternative procedure is described for the preparation of single cells of GM14667 and GM05423 cell lines to use as reference DNA and for sex-mismatched control experiments. A guide is also provided for micromanipulation of single cells. Next, the unit describes whole-genome amplification using adapter-linker PCR (Ampli1 WGA Kit) and an alternative nonlinear WGA method (PicoPLEX WGA Kit) for single-cell amplification. A protocol is also included for reamplification of Ampli1 WGA products, which can be used for aCGH as well. Finally, the use of 4 × 180k oligonucleotide microarrays to perform aCGH with single-cell WGA products is described.

New tools for embryo selection: comprehensive chromosome screening by array comparative genomic hybridization.

The objective of this study was to evaluate the usefulness of comprehensive chromosome screening (CCS) using array comparative genomic hybridization (aCGH). The study included 1420 CCS cycles for recurrent miscarriage (n = 203); repetitive implantation failure (n = 188); severe male factor (n = 116); previous trisomic pregnancy (n = 33); and advanced maternal age (n = 880). CCS was performed in cycles with fresh oocytes and embryos (n = 774); mixed cycles with fresh and vitrified oocytes (n = 320); mixed cycles with fresh and vitrified day-2 embryos (n = 235); and mixed cycles with fresh and vitrified day-3 embryos (n = 91). Day-3 embryo biopsy was performed and analyzed by aCGH followed by day-5 embryo transfer. Consistent implantation (range: 40.5-54.2%) and pregnancy rates per transfer (range: 46.0-62.9%) were obtained for all the indications and independently of the origin of the oocytes or embryos. However, a lower delivery rate per cycle was achieved in women aged over 40 years (18.1%) due to the higher percentage of aneuploid embryos (85.3%) and lower number of cycles with at least one euploid embryo available per transfer (40.3%). We concluded that aneuploidy is one of the major factors which affect embryo implantation.

Functional performance of aCGH design for clinical cytogenetics.

Array-comparative genomic hybridization (aCGH) technology enables rapid, high-resolution analysis of genomic rearrangements. With the use of it, genome copy number changes and rearrangement breakpoints can be detected and analyzed at resolutions down to a few kilobases. An exon array CGH approach proposed recently accurately measures copy-number changes of individual exons in the human genome. The crucial and highly non-trivial starting task is the design of an array, i.e. the choice of appropriate (multi)set of oligos. The success of the whole high-level analysis depends on the quality of the design. Also, the comparison of several alternative designs of array CGH constitutes an important step in development of new diagnostic chip. In this paper, we deal with these two often neglected issues. We propose a new approach to measure the quality of array CGH designs. Our measures reflect the robustness of rearrangements detection to the noise (mostly experimental measurement error). The method is parametrized by the segmentation algorithm used to identify aberrations. We implemented the efficient Monte Carlo method for testing noise robustness within DNAcopy procedure. Developed framework has been applied to evaluation of functional quality of several optimized array designs.

Prenatal diagnosis of ring chromosome 2 with lissencephaly and 2p25.3 and 2q37.3 microdeletions detected using array comparative genomic hybridization.

We present rapid aneuploidy diagnosis of ring chromosome 2 with 2p25.3 and 2q37.3 microdeletions by aCGH using uncultured amniocytes in a fetus with IUGR, microcephaly, lissencephaly and ambiguous external genitalia. Our case adds lissencephaly to the list of CNS abnormalities in ring chromosome 2 with 2p25.3 and 2q37.3 microdeletions. We discuss the consequence of haploinsufficiency of HDAC4, KIF1A, PASK, HDLBP, FRAP2 and D2HGDH on 2q37.3, and haploinsufficiency of MYT1L, SNTG2 and TPO on 2p25.3 in this case.

Ultradense array CGH and discovery of micro-copy number alterations and gene fusions in the cancer genome.

The characterization of molecular alterations specific to cancer facilitates the discovery of predictive and prognostic biomarkers important to targeted therapeutics. Alterations critical to cancer therapeutics include copy number alterations (CNAs) such as gene amplifications and deletions as well as genomic rearrangements resulting in gene fusions. There are two genome-wide technologies used to detect CNAs: next generation sequencing (NGS) and dense microarray based comparative genomic hybridization, termed array CGH (aCGH). aCGH is a mature robust technology of lower cost and more accessible than NGS. This chapter describes the protocol steps and analysis required to obtain reliable aCGH results from clinical samples. Technical options and various necessary compromises related to the nature of clinical material are considered and the consequences of these choices for data analysis and interpretation are discussed. The chapter includes brief description of the data analysis, even though analysis is often performed by bioinformaticians. Today's cancer research requires collaboration of clinicians, molecular biologists, and mathematicians. Acquaintance with the basic principles related to the extraction of the data from arrays, its normalization and the algorithms available for analysis provides a baseline for mutual understanding and communication.

The use of cytogenetic microarrays in myelodysplastic syndrome characterization.

Various microarray platforms, including BAC, oligonucleotide, and SNP arrays, have been shown to -provide clinically useful diagnostic and prognostic information for patients with myelodysplastic syndromes (MDS). Clinically useful arrays are designed with specific purposes in mind and with attention to genomic content and probe density. All array types have been shown to detect genomic copy gains and losses, with SNP arrays having the added advantage of detecting copy neutral loss of heterozygosity (CNLOH). The finding of CNLOH has led to the identification of certain disease genes implicated in the initiation or progression of myeloid diseases. In addition, SNP karyotyping alone, or in conjunction with routine cytogenetics, can affect the outcome prediction and improve prognostic stratification of patients with MDS. Patients who were reclassified after array testing as having adverse-risk chromosomal findings correlated with poor survival. Results of over 25 published studies support the use of arrays in MDS testing. Because few balanced translocations are found in MDS, this disease is particularly amenable to microarray testing, and studies have shown better disease classification, identification of cryptic changes, and prognostication in this heterogeneous group of disorders. Novel genomic alterations identified by array testing may lead to better targeted therapies for treating patients with MDS.

High quality genomic copy number data from archival formalin-fixed paraffin-embedded leiomyosarcoma: optimisation of universal linkage system labelling.

Most soft tissue sarcomas are characterized by genetic instability and frequent genomic copy number aberrations that are not subtype-specific. Oligonucleotide microarray-based Comparative Genomic Hybridisation (array CGH) is an important technique used to map genome-wide copy number aberrations, but the traditional requirement for high-quality DNA typically obtained from fresh tissue has limited its use in sarcomas. Although large archives of Formalin-fixed Paraffin-embedded (FFPE) tumour samples are available for research, the degradative effects of formalin on DNA from these tissues has made labelling and analysis by array CGH technically challenging. The Universal Linkage System (ULS) may be used for a one-step chemical labelling of such degraded DNA. We have optimised the ULS labelling protocol to perform aCGH on archived FFPE leiomyosarcoma tissues using the 180k Agilent platform. Preservation age of samples ranged from a few months to seventeen years and the DNA showed a wide range of degradation (when visualised on agarose gels). Consistently high DNA labelling efficiency and low microarray probe-to-probe variation (as measured by the derivative log ratio spread) was seen. Comparison of paired fresh and FFPE samples from identical tumours showed good correlation of CNAs detected. Furthermore, the ability to macro-dissect FFPE samples permitted the detection of CNAs that were masked in fresh tissue. Aberrations were visually confirmed using Fluorescence in situ Hybridisation. These results suggest that archival FFPE tissue, with its relative abundance and attendant clinical data may be used for effective mapping for genomic copy number aberrations in such rare tumours as leiomyosarcoma and potentially unravel clues to tumour origins, progression and ultimately, targeted treatment.

Challenges of interpreting copy number variation in syndromic and non-syndromic congenital heart defects.

Array comparative genomic hybridization (aCGH) has led to an increased detection of causal chromosomal imbalances in individuals with congenital heart defects (CHD). The introduction of aCGH as a diagnostic tool in a clinical cardiogenetic setting entails numerous challenges. Based on our own experience as well as those of others described in the literature, we outline the state of the art and attempt to answer a number of outstanding questions such as the detection frequency of causal imbalances in different patient populations, the added value of higher-resolution arrays, and the existence of predictive factors in syndromic cases. We introduce a step-by-step approach for clinical interpretation of copy number variants (CNV) detected in CHD, which is primarily based on gene content and overlap with known chromosomal syndromes, rather than on CNV inheritance and size. Based on this algorithm, we have reclassified the detected aberrations in aCGH studies for their causality for syndromic and non-syndromic CHD. From this literature overview, supplemented with own investigations in a cohort of 46 sporadic patients with severe non-syndromic CHD, it seems clear that the frequency of causal CNVs in non-syndromic CHD populations is lower than that in syndromic CNV populations (3.6 vs. 19%). Moreover, causal CNVs in non-syndromic CHD mostly involve imbalances with a moderate effect size and reduced penetrance, whereas the majority of causal imbalances in syndromic CHD consistently affects human development and significantly reduces reproductive fitness.

Microarray-based CGH and copy number analysis of FFPE samples.

Over the past decade, utilization of microarray technology has flourished in biomedical research. It has evolved rapidly into a revolutionary tool that offers deeper insight into the molecular basis associated with complex diseases, especially in the field of cancer. Specifically, array-based Comparative Genomic Hybridization (aCGH) permits the detection of genome-wide copy number alterations with high resolution. Microarray application to DNA extracted from formalin-fixed paraffin-embedded tissue (FFPE), in particular, poses a challenge due to the partially degraded nature and compromised quality of the DNA. This chapter gives a description of the several CGH-microarray platforms currently available and offers practical steps that guide you through optimal handling and superior aCGH data acquisition of DNA extracted from FFPE tissues.

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

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.

Detection of mosaicism for genome imbalance in a cohort of 3,042 clinical cases using an oligonucleotide array CGH platform.

Mosaicism for chromosome imbalance has traditionally been detected by karyotype analysis. The introduction of array CGH into clinical diagnostic laboratories and routine clinical practice has raised concerns as to the ability of this new test to detect the presence of more than one cell line. We present our validation data on the detection of chromosome mosaicism by oligonucleotide array CGH, and the cases detected in a cohort of 3042 clinical referrals. Using an artificial mosaicism series, we found that oligonucleotide array CGH using specific analysis parameters could accurately measure levels of mosaicism down to 10% and that the degree of mosaicism could be predicted from fluorescence ratios. We detected 12 cases of mosaicism in our clinical cohort, in 9 of which there was no previous indication of mosaicism. In two cases, G-banded chromosome analysis had been carried out previously, and had failed to detect the abnormal cell line. Three cases had mosaicism for the X chromosome and 9 involved autosomes, of which 4 were mosaic for whole chromosome trisomies, one for whole chromosome monosomy, and four were mosaic for segmental imbalances. We conclude that oligonucleotide array CGH has the power to detect a range of mosaic abnormalities in clinical diagnostic samples.

The assessment of array comparative genomic hybridization in complex karyotype analyses.

Molecular-cytogenetic methods were used to analyse and specify complex genome rearrangements in malignant cells. Twelve samples of bone marrow cells were collected from patients with myelodysplastic syndromes (MDS). The complex karyotypes were examined by multicolour fluorescence in situ hybridization (mFISH), high-resolution multicolour banding (mBAND) and array comparative genomic hybridization (aCGH). For aCGH, DNA was isolated from fixed bone marrow cells in methanol and acetic acid and amplified by whole-genome amplification. Three samples were analysed by the oligonucleotide array NimbleGen on the basis of full service. BAC-based Haematochips (BlueGnome) were used for the other nine samples. Sensitivity and detection limits of both methods were compared. The results obtained by mFISH/mBAND were in most cases confirmed by the microarray technique. aCGH detected 43 unbalanced chromosomal changes that were also identified by classical cytogenetics and FISH. Moreover, aCGH discovered 14 additional changes. Cryptic amplifications and deletions were characterized with a resolution of 0.5 Mb. In one bone marrow sample with suspected monosomy 5 detected by conventional cytogenetic analysis, aCGH revealed a 22.3 Mb region of chromosome 5 inserted in another autosome within the complex karyotype. Amplified DNA was successfully used for aCGH in 11 out of 12 cases, improving resolution of unbalanced chromosomal aberrations. The combination of both approaches brought more detailed description of complex karyotypes and is highly recommended.

Genome-wide oligonucleotide array comparative genomic hybridization for etiological diagnosis of mental retardation: a multicenter experience of 1499 clinical cases.

To assess the clinical utility of genome-wide oligonucleotide arrays in diagnosis of mental retardation and to address issues relating to interpretation of copy number changes (CNCs), we collected results on a total of 1499 proband patients from five academic diagnostic laboratories where the same 44K array platform has been used. Three of the five laboratories achieved a diagnostic yield of 14% and the other two had a yield of 11 and 7%, respectively. Approximately 80% of the abnormal cases had a single segment deletion or duplication, whereas the remaining 20% had a compound genomic imbalance involving two or more DNA segments. Deletion of 16p11.2 is a common microdeletion syndrome associated with mental retardation. We classified pathogenic CNCs into six groups according to the structural changes. Our data have demonstrated that the 44K platform provides a reasonable resolution for clinical use and a size of 300 kb can be used as a practical cutoff for further investigations of the clinical relevance of a CNC detected with this platform. We have discussed in depth the issues associated with the clinical use of array CGH and provided guidance for interpretation, reporting, and counseling of test results based on our experience.

SnoopCGH: software for visualizing comparative genomic hybridization data.

UNLABELLED: Array-based comparative genomic hybridization (CGH) technology is used to discover and validate genomic structural variation, including copy number variants, insertions, deletions and other structural variants (SVs). The visualization and summarization of the array CGH data outputs, potentially across many samples, is an important process in the identification and analysis of SVs. We have developed a software tool for SV analysis using data from array CGH technologies, which is also amenable to short-read sequence data. AVAILABILITY AND IMPLEMENTATION: SnoopCGH is written in java and is available from http://snoopcgh.sourceforge.net/

Comparative genomic hybridization on BAC arrays.

Alterations in genomic DNA are a key feature of many constitutional disorders and cancer. The discovery of the underlying regions of gene dosage has thus been essential in dissecting complex disease phenotypes and identifying targets for therapeutic intervention and diagnostic testing. The development of array comparative genomic hybridization (aCGH) using bacterial artificial chromosomes (BACs) as hybridization targets has facilitated the discovery and fine mapping of novel genomic alterations allowing rapid identification of target genes. In BAC aCGH, DNA samples are first labeled with fluorescent dyes through a random priming reaction with 100-400 ng of genomic DNA. This probe is then co-hybridized to an array consisting of BAC clones, either tiling the genome (approximately 50 kbp resolution) or spaced at intervals (e.g., 1 Mbp resolution). The resulting arrays are then imaged and the signal at each locus is compared between a reference and test sample to determine the copy number status. The DNA samples to be analyzed may be derived from either fresh, frozen, or formalin-fixed paraffin-embedded material, and sample requirements are currently significantly lower than those for oligonucleotide platforms due to the high probe-binding capacity of BAC clone targets (approximately 150 kbp) compared to oligonucleotides (25-80 bp). In this chapter, we describe in detail the technical procedure required to perform copy number analysis of genomes with BAC aCGH.

Comparative genomic hybridization on spotted oligonucleotide microarrays.

Recent advances in DNA microarray technology have enabled researchers to comprehensively characterize the complex genomes of higher eukaryotic organisms at an unprecedented level of detail. Array-based comparative genomic hybridization (Array-CGH) has been widely used for detecting DNA copy number alterations on a genomic scale, where the mapping resolution is limited only by the number of probes on the DNA microarray. In this chapter, we present a validated protocol utilizing print-tip spotted HEEBO (Human Exonic Evidence Based Oligonucleotide) microarrays for conducting array-CGH using as little as 25 ng of genomic DNA from a wide variety of sources, including cultured cell lines and clinical specimens, with high spatial resolution and array-to-array reproducibility.

Comparative genomic hybridization by representational oligonucleotide microarray analysis.

The central cause to any cancer ultimately lies in the genome and the initial alterations that result in changes in gene expression that are reflected in the phenotype of the cancer cell. The gene expression data are rich in information but the primary lesions responsible for carcinogenesis are obscured due to the complex cascade of expression changes that can occur. The primary lesions can be characterized by the smallest of point mutations to small insertions and deletions (in/dels) to much larger deletions and amplifications (for simplicity all copy number gains will be referred to as amplifications) as well as balanced or unbalanced translocations. In addition to these mutations there are a myriad of epigenetic alterations that affect the cells phenotype. Any gene if important to tumor growth will be altered by mutation or by deletion/amplification eventually, and if a large number of tumor samples is analyzed the majority of these genes will be detected. This chapter describes a variation of comparative genomic hybridization, called Representational oligonucleotide microarray analysis (ROMA), that surveys reduced-complexity representations of tumor genomic DNA to discover deletions and amplifications (and the underlying cancer genes).