Der Stellenwert der ?array comparative genomic hybridization“ in der Pr?nataldiagnostik

Array comparative genomic hybridization (CGH) enables, without the need for cell culture, the detection of changes in copy numbers with high accuracy below the resolution of standard chromosome analysis. The implementation of array?CGH in prenatal diagnosis has been hesitant in spite of these obvious advantages. This has been predominantly due to the likelihood of finding copy number variations (CNVs) of uncertain clinical significance. In prenatal diagnosis array?CGH should not be offered as a first tier but as an adjunct to standard diagnostic procedures in order to minimize uncertainty. Indications for the use of array?CGH will be defined and substantiated in the present article. Guidelines should be established at each laboratory regarding the minimum size of CNVs to be assessed and the genomic regions considered clinically significant.     

Reference-unbiased copy number variant analysis using CGH microarrays

Comparative genomic hybridization (CGH) microarrays have been used to determine copy number variations (CNVs) and their effects on complex diseases. Detection of absolute CNVs independent of genomic variants of an arbitrary reference sample has been a critical issue in CGH array experiments. Whole genome analysis using massively parallel sequencing with multiple ultra-high resolution CGH arrays provides an opportunity to catalog highly accurate genomic variants of the reference DNA (NA10851). Using information on variants, we developed a new method, the CGH array reference-free algorithm (CARA), which can determine reference-unbiased absolute CNVs from any CGH array platform. The algorithm enables the removal and rescue of false positive and false negative CNVs, respectively, which appear due to the effects of genomic variants of the reference sample in raw CGH array experiments. We found that the CARA remarkably enhanced the accuracy of CGH array in determining absolute CNVs. Our method thus provides a new approach to interpret CGH array data for personalized medicine.     

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.     

Appropriateness of array‐CGH in the ADHD clinics: A comparative study

Attention deficit hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorder with a worldwide prevalence of about 5%. The disorder is characterized by inattentive, hyperactive and impulsive behavior and is often comorbid with other neuropsychiatric conditions. Array comparative genomic hybridization (array‐CGH) testing has been proved to be useful to detect chromosomal aberrations in several neuropsychiatric conditions including autism spectrum disorders (ASD) and intellectual disability (ID). The usefulness of array‐CGH in the ADHD clinics is still debated and no conclusive evidence has been reached to date. We performed array‐CGH in 98 children and adolescents divided in two similarly sized groups according to the clinical diagnosis: (a) one group diagnosed with ADHD as primary diagnosis; (b) the other group in which ADHD was co‐morbid with ASD and/or ID. We detected pathogenetic and likely pathogenetic copy number variants (CNVs) in 12% subjects in which ADHD was co‐morbid with autism and/or intellectual disability and in 8.5% subjects diagnosed with ADHD as primary diagnosis. Detection of CNVs of unknown clinical significance was similar in the two groups being 27% and 32%, respectively. Benign and likely benign CNVs accounted for 61% and 59.5% in the first and second group, respectively. Differences in the diagnostic yield were not statistically significant between the two groups (P > .05). Our data strongly suggest that array‐CGH (a) is a valuable diagnostic tool to detect clinically significant CNVs in individuals with ADHD even in the absence of comorbidity with ASD and/or ID and (b) should be implemented routinely in the ADHD clinics.     

Array CGH analysis of a cohort of Russian patients with intellectual disability

The use of array comparative genomic hybridization (array CGH) as a diagnostic tool in molecular genetics has facilitated the identification of many new microdeletion/microduplication syndromes (MMSs). Furthermore, this method has allowed for the identification of copy number variations (CNVs) whose pathogenic role has yet to be uncovered. Here, we report on our application of array CGH for the identification of pathogenic CNVs in 79 Russian children with intellectual disability (ID). Twenty-six pathogenic or likely pathogenic changes in copy number were detected in 22 patients (28%): 8 CNVs corresponded to known MMSs, and 17 were not associated with previously described syndromes. In this report, we describe our findings and comment on genes potentially associated with ID that are located within the CNV regions.     

Comparative genomic hybridization arrays in clinical pathology: progress and challenges

Array-based comparative genomic hybridization (array CGH) genome scanning is a powerful method for the global detection of gains and losses of genetic material in both congenital and neoplastic disorders. When used as a clinical diagnostic test, array CGH combines the whole genome perspective of traditional G-banded cytogenetics with the targeted identification of cryptic chromosomal abnormalities characteristic of fluorescence in situ hybridization (FISH). However, the presence of structural variants in the human genome can complicate analysis of patient samples, and array CGH does not provide morphologic information about chromosome structure, balanced translocations, or the actual chromosomal location of segmental duplications. Identification of such anomalies has significant diagnostic and prognostic implications for the patient. We therefore propose that array CGH should be used as a guide to the presence of genomic structural rearrangements in germline and tumor genomes that can then be further characterized by FISH or G-banding, depending on the clinical scenario. In this article, we share some of our experiences with diagnostic array CGH and discuss recent progress and challenges involved with the integration of array CGH into clinical laboratory medicine.     

Duplication of CXC chemokine genes on chromosome 4q13 in a melanoma-prone family

Copy number variations (CNVs) have been shown to contribute substantially to disease susceptibility in several inherited diseases including cancer. We conducted a genome-wide search for CNVs in blood-derived DNA from 79 individuals (62 melanoma patients and 17 spouse controls) of 30 high-risk melanoma-prone families without known segregating mutations using genome-wide comparative genomic hybridization (CGH) tiling arrays. We identified a duplicated region on chromosome 4q13 in germline DNA of all melanoma patients in a melanoma-prone family with three affected siblings. We confirmed the duplication using quantitative PCR and a custom-made CGH array design spanning the 4q13 region. The duplicated region contains 10 genes, most of which encode CXC chemokines. Among them, CXCL1 (melanoma growth-stimulating activity α) and IL8 (interleukin 8) have been shown to stimulate melanoma growth in vitro and in vivo. Our data suggest that the alteration of CXC chemokine genes may confer susceptibility to melanoma.     

Identification of copy number variants on human chromosome 22 in patients with a variety of clinical findings

The aims of this study were to create a copy number variant (CNV) profile of human chromosome 22 and to establish a genotype-phenotype correlation for patients with genomic abnormalities on chromosome 22. Thus, 1,654 consecutive pediatric patients with a diversity of clinical findings were evaluated by high-resolution chromosomal microarray analysis (CMA). We identified 25 individuals with abnormal CNVs on chromosome 22, representing 1.5% of the cases analyzed in this cohort. Meanwhile, we detected 1,298 benign CNVs on this chromosome in these individuals. Twenty-one of the 25 abnormal CNVs and the majority of the benign CNVs occurred through involvement of the 8 unstable genomic regions enriched with low copy repeats (LCR22A-H). The highly dynamic status of LCR22s within the 22q11 region facilitates the formation of diverse genomic abnormalities. This CNV profile provides a general perspective of the spectrum of chromosome 22 genomic imbalances and subsequently improves the CNV-phenotype correlations.     

A Genome-Wide Analysis of Array-Based Comparative Genomic Hybridization (CGH) Data to Detect Intra-Species Variations and Evolutionary Relationships

Array-based comparative genomics hybridization (aCGH) has gained prevalence as an effective technique for measuring structural variations in the genome. Copy-number variations (CNVs) form a large source of genomic structural variation, but it is not known whether phenotypic differences between intra-species groups, such as divergent human populations, or breeds of a domestic animal, can be attributed to CNVs. Several computational methods have been proposed to improve the detection of CNVs from array CGH data, but few population studies have used CGH data for identification of intra-species differences. In this paper we propose a novel method of genome-wide comparison and classification using CGH data that condenses whole genome information, aimed at quantification of intra-species variations and discovery of shared ancestry. Our strategy included smoothing CGH data using an appropriate denoising algorithm, extracting features via wavelets, quantifying the information via wavelet power spectrum and hierarchical clustering of the resultant profile. To evaluate the classification efficiency of our method, we used simulated data sets. We applied it to aCGH data from human and bovine individuals and showed that it successfully detects existing intra-specific variations with additional evolutionary implications.     

Molekulare Karyotypisierung in der klinischen Anwendung

Microarray technology for the detection of putative pathological submicroscopic copy number variants (CNV) has become a standard tool in the field of molecular cytogenetics in recent years. In addition to the identification of somatic CNVs in tumour genetics this technology is increasingly used for the analysis of constitutional CNVs in patients with developmental delay. Array-based genomic hybridisation increases sensitivity in comparison to more conventional technologies such as comparative genomic hybridisation (CGH). Recent developments now allow a genome-wide detection of submicroscopic chromosomal alterations, deletions and duplications smaller than 100 Kb, thus significantly increasing the detection rate of chromosomal aberrations in patients suffering from idiopathic mental retardation. Several centers are already using array technology in their routine setting in the diagnostic approach to syndromes. Therefore, this overview focuses on the similarities, as well as the differences, of several basic array techniques.     

Genome-wide mapping of copy number variation in humans: comparative analysis of high resolution array platforms

Accurate and efficient genome-wide detection of copy number variants (CNVs) is essential for understanding human genomic variation, genome-wide CNV association type studies, cytogenetics research and diagnostics, and independent validation of CNVs identified from sequencing based technologies. Numerous, array-based platforms for CNV detection exist utilizing array Comparative Genome Hybridization (aCGH), Single Nucleotide Polymorphism (SNP) genotyping or both. We have quantitatively assessed the abilities of twelve leading genome-wide CNV detection platforms to accurately detect Gold Standard sets of CNVs in the genome of HapMap CEU sample NA12878, and found significant differences in performance. The technologies analyzed were the NimbleGen 4.2 M, 2.1 M and 3×720 K Whole Genome and CNV focused arrays, the Agilent 1×1 M CGH and High Resolution and 2×400 K CNV and SNP+CGH arrays, the Illumina Human Omni1Quad array and the Affymetrix SNP 6.0 array. The Gold Standards used were a 1000 Genomes Project sequencing-based set of 3997 validated CNVs and an ultra high-resolution aCGH-based set of 756 validated CNVs. We found that sensitivity, total number, size range and breakpoint resolution of CNV calls were highest for CNV focused arrays. Our results are important for cost effective CNV detection and validation for both basic and clinical applications.     

High-throughput analysis of subtelomeric chromosome rearrangements by use of array-based comparative genomic hybridization

Telomeric chromosome rearrangements may cause mental retardation, congenital anomalies, and miscarriages. Automated detection of subtle deletions or duplications involving telomeres is essential for high-throughput diagnosis, but impossible when conventional cytogenetic methods are used. Array-based comparative genomic hybridization (CGH) allows high-resolution screening of copy number abnormalities by hybridizing differentially labeled test and reference genomes to arrays of robotically spotted clones. To assess the applicability of this technique in the diagnosis of (sub)telomeric imbalances, we here describe a blinded study, in which DNA from 20 patients with known cytogenetic abnormalities involving one or more telomeres was hybridized to an array containing a validated set of human-chromosome-specific (sub)telomere probes. Single-copy-number gains and losses were accurately detected on these arrays, and an excellent concordance between the original cytogenetic diagnosis and the array-based CGH diagnosis was obtained by use of a single hybridization. In addition to the previously identified cytogenetic changes, array-based CGH revealed additional telomere rearrangements in 3 of the 20 patients studied. The robustness and simplicity of this array-based telomere copy-number screening make it highly suited for introduction into the clinic as a rapid and sensitive automated diagnostic procedure.     

Allele-specific disparity in breast cancer

Background

Molecular alterations critical to development of cancer include mutations, copy number alterations (amplifications and deletions) as well as genomic rearrangements resulting in gene fusions. Massively parallel next generation sequencing, which enables the discovery of such changes, uses considerable quantities of genomic DNA (> 5 ug), a serious limitation in ever smaller clinical samples. However, a commonly available microarray platforms such as array comparative genomic hybridization (array CGH) allows the characterization of gene copy number at a single gene resolution using much smaller amounts of genomic DNA. In this study we evaluate the sensitivity of ultra-dense array CGH platforms developed by Agilent, especially that of the 1 million probe array (1 M array), and their application when whole genome amplification is required because of limited sample quantities.

Methods

We performed array CGH on whole genome amplified and not amplified genomic DNA from MCF-7 breast cancer cells, using 244 K and 1 M Agilent arrays. The ADM-2 algorithm was used to identify micro-copy number alterations that measured less than 1 Mb in genomic length.

Results

DNA from MCF-7 breast cancer cells was analyzed for micro-copy number alterations, defined as measuring less than 1 Mb in genomic length. The 4-fold extra resolution of the 1 M array platform relative to the less dense 244 K array platform, led to the improved detection of copy number variations (CNVs) and micro-CNAs. The identification of intra-genic breakpoints in areas of DNA copy number gain signaled the possible presence of gene fusion events. However, the ultra-dense platforms, especially the densest 1 M array, detect artifacts inherent to whole genome amplification and should be used only with non-amplified DNA samples.

Conclusions

This is a first report using 1 M array CGH for the discovery of cancer genes and biomarkers. We show the remarkable capacity of this technology to discover CNVs, micro-copy number alterations and even gene fusions. However, these platforms require excellent genomic DNA quality and do not tolerate relatively small imperfections related to the whole genome amplification.     

Interpretation of array comparative genome hybridization data: a major challenge

The advent and application of high-resolution array-based comparative genome hybridization (array CGH) has led to the detection of large numbers of copy number variants (CNVs) in patients with developmental delay and/or multiple congenital anomalies as well as in healthy individuals. The notion that CNVs are also abundantly present in the normal population challenges the interpretation of the clinical significance of detected CNVs in patients. In this review we will illustrate a general clinical workflow based on our own experience that can be used in routine diagnostics for the interpretation of CNVs.     

Comparative genomic hybridization-array analysis enhances the detection of aneuploidies and submicroscopic imbalances in spontaneous miscarriages

Miscarriage is a condition that affects 10%-15% of all clinically recognized pregnancies, most of which occur in the first trimester. Approximately 50% of first-trimester miscarriages result from fetal chromosome abnormalities. Currently, G-banded chromosome analysis is used to determine if large-scale genetic imbalances are the cause of these pregnancy losses. This technique relies on the culture of cells derived from the fetus, a technique that has many limitations, including a high rate of culture failure, maternal overgrowth of fetal cells, and poor chromosome morphology. Comparative genomic hybridization (CGH)-array analysis is a powerful new molecular cytogenetic technique that allows genomewide analysis of DNA copy number. By hybridizing patient DNA and normal reference DNA to arrays of genomic clones, unbalanced gains or losses of genetic material across the genome can be detected. In this study, 41 product-of-conception (POC) samples, which were previously analyzed by G-banding, were tested using CGH arrays to determine not only if the array could identify all reported abnormalities, but also whether any previously undetected genomic imbalances would be discovered. The array methodology detected all abnormalities as reported by G-banding analysis and revealed new abnormalities in 4/41 (9.8%) cases. Of those, one trisomy 21 POC was also mosaic for trisomy 20, one had a duplication of the 10q telomere region, one had an interstitial deletion of chromosome 9p, and the fourth had an interstitial duplication of the Prader-Willi/Angelman syndrome region on chromosome 15q, which, if maternally inherited, has been implicated in autism. This retrospective study demonstrates that the DNA-based CGH-array technology overcomes many of the limitations of routine cytogenetic analysis of POC samples while enhancing the detection of fetal chromosome aberrations.     

Chromosome 15q13.3 microduplications are associated with treatment refractory major depressive disorder

Major depressive disorder (MDD) affects approximately 15 million Americans. Approximately 2 million of these are classified as being refractory to treatment (TR‐MDD). Because of the lack of available therapies for TR‐MDD, and the high risk of suicide, there is interest in identifying new treatment modalities and diagnostic methods. Understanding of the impact of genomic copy number variation in the etiology of a variety of neuropsychiatric phenotypes is increasing. Low copy repeat elements at 15q13.3 facilitate non‐allelic homologous recombination, resulting in recurrent copy number variants (CNVs). Numerous reports have described association between microdeletions in this region and a variety of neuropsychiatric phenotypes, with CHRNA7 implicated as a candidate gene. However, the pathogenicity of 15q13.3 duplications is less clear. As part of an ongoing study, in which we have identified a number of metabolomic anomalies in spinal fluid from TR‐MDD patients, we also evaluated genomic copy number variation in patients (n = 125) and controls (n = 26) via array‐based copy number genomic hybridization (CGH); the case frequency was compared with frequencies reported in a prior study as well as a larger population‐sized cohort. We identified five TR‐MDD patients with microduplications involving CHRNA7. CHRNA7 duplications are the most common CNVs identified by clinical CGH in this cohort. Therefore, this study provides insight into the potential involvement of CHRNA7 duplications in the etiology of TR‐MDD and informs those involved with care of affected individuals.     

Outcome of array CGH analysis for 255 subjects with intellectual disability and search for candidate genes using bioinformatics

Array CGH enables the detection of pathogenic copy number variants (CNVs) in 5–15% of individuals with intellectual disability (ID), making it a promising tool for uncovering ID candidate genes. However, most CNVs encompass multiple genes, making it difficult to identify key disease gene(s) underlying ID etiology. Using array CGH we identified 47 previously unreported unique CNVs in 45/255 probands. We prioritized ID candidate genes using five bioinformatic gene prioritization web tools. Gene priority lists were created by comparing integral genes from each CNV from our ID cohort with sets of training genes specific either to ID or randomly selected. Our findings suggest that different training sets alter gene prioritization only moderately; however, only the ID gene training set resulted in significant enrichment of genes with nervous system function (19%) in prioritized versus non-prioritized genes from the same de novo CNVs (7%, p < 0.05). This enrichment further increased to 31% when the five web tools were used in concert and included genes within mitogen-activated protein kinase (MAPK) and neuroactive ligand-receptor interaction pathways. Gene prioritization web tools enrich for genes with relevant function in ID and more readily facilitate the selection of ID candidate genes for functional studies, particularly for large CNVs.     

Copy number variation in Fayoumi and Leghorn chickens analyzed using array comparative genomic hybridization

Copy number variation refers to regions along chromosomes that harbor a type of structural variation, such as duplications or deletions. Copy number variants (CNVs) play a role in many important traits as well as in genetic diversity. Previous analyses of chickens using array comparative genomic hybridizations or single‐nucleotide polymorphism chip assays have been performed on various breeds and genetic lines to discover CNVs. In this study, we assessed individuals from two highly inbred (inbreeding coefficiency > 99.99%) lines, Leghorn G‐B2 and Fayoumi M15.2, to discover novel CNVs in chickens. These lines have been previously studied for disease resistance, and to our knowledge, this represents the first global assessment of CNVs in the Fayoumi breed. Genomic DNA from individuals was examined using the Agilent chicken 244 K comparative genomic hybridization array and quantitative PCR. We identified a total of 273 CNVs overall, with 112 CNVs being novel and not previously reported. Quantitative PCR using the standard curve method validated a subset of our array data. Through enrichment analysis of genes within CNV regions, we observed multiple chromosomes, terms and pathways that were significantly enriched, largely dealing with the major histocompatibility complex and immune responsiveness. Using an additional round of computational and statistical analysis with a different bioinformatic pipeline, we identified 43 CNVs among these as high‐confidence regions, 14 of which were found to be novel. We further compared and contrasted individuals of the two inbred lines to discover regions that have a significant difference in copy number between lines. A total of 40 regions had significant deletions or duplications between the lines. Gene Ontology analysis of genomic regions containing CNVs between lines also was performed. This between‐line candidate CNV list will be useful in studies with these two unique genetic lines, which may harbor variations that underlie quantitative trait loci for disease resistance and other important traits. Through the global discovery of novel CNVs in chicken, these data also provide resources for further genetic and functional genomics studies.     

Designing a simple multiplex ligation-dependent probe amplification （MLPA） assay for rapid detection of copy number variants in the genome

Copy number variants （CNVs） are pervasive in the human genome and are responsible for many Mendelian diseases and genomic disorders. The detection of CNVs is an essential element of a complete mutation screening strategy. Many techniques have been developed for gene dosage testing. Multiplex ligation-dependent probe amplification （MLPA） is a robust, easy and flexible technique that can detect both deletions and duplications for more than 40 loci in one assay. It has been widely used in research and diagnostic laboratories. We routinely develop our own MLPA assays for quick validation of array comparative genomic hybridization （CGH） findings. Here we discuss the general principles and critical aspects of MLPA assay development and validation using all synthetic MLPA probes. We believe that MLPA will play important roles in the rapid detection of genomic disorders associated with genomic imbalances, the confirmation of pathogenic mutations involving exonic deletions/duplications, CNV genotyping and population frequency analysis of CNVs.     

A method for calling gains and losses in array CGH data

Array CGH is a powerful technique for genomic studies of cancer. It enables one to carry out genome-wide screening for regions of genetic alterations, such as chromosome gains and losses, or localized amplifications and deletions. In this paper, we propose a new algorithm 'Cluster along chromosomes' (CLAC) for the analysis of array CGH data. CLAC builds hierarchical clustering-style trees along each chromosome arm (or chromosome), and then selects the 'interesting' clusters by controlling the False Discovery Rate (FDR) at a certain level. In addition, it provides a consensus summary across a set of arrays, as well as an estimate of the corresponding FDR. We illustrate the method using an application of CLAC on a lung cancer microarray CGH data set as well as a BAC array CGH data set of aneuploid cell strains.