CGHPRO – A comprehensive data analysis tool for array CGH

Background  

Array CGH (Comparative Genomic Hybridisation) is a molecular cytogenetic technique for the genome wide detection of chromosomal imbalances. It is based on the co-hybridisation of differentially labelled test and reference DNA onto arrays of genomic BAC clones, cDNAs or oligonucleotides, and after correction for various intervening variables, loss or gain in the test DNA can be indicated from spots showing aberrant signal intensity ratios.     

Comparative Genomic Hybridization As a New Method for Detection of Genomic Imbalance

Comparative Genomic Hybridization (CGH) is a molecular cytogenetic method for detecting chromosomal imbalances by comparing the copy number of DNA sequences in cells of tested tissue and the reference specimen. CGH is based on two-color fluorescence suppressive in situ hybridization of genomic test and reference DNAs, each labeled with a different fluorochrome, to metaphase chromosomes of a healthy individual. First described by Kallioniemi et al. in 1992, the CGH assay has been widely used for identification and characterization of both numerical and unbalanced structural chromosome abnormalities in cells of different tissues at various pathological conditions in humans, especially in tumor diseases. We discuss the specific features and quality control of comparative genomic hybridization, its advantages and limitations in detection of genomic imbalance and the prospects for development of this technology.     

Genomic characterization of some Iranian children with idiopathic mental retardation using array comparative genomic hybridization

BACKGROUND:

Mental retardation (MR) has a prevalence of 1-3% and genetic causes are present in more than 50% of patients. Chromosomal abnormalities are one of the most common genetic causes of MR and are responsible for 4-28% of mental retardation. However, the smallest loss or gain of material visible by standard cytogenetic is about 4 Mb and for smaller abnormalities, molecular cytogenetic techniques such as array comparative genomic hybridization (array CGH) should be used. It has been shown that 15-25% of idiopathic MR (IMR) has submicroscopic rearrangements detectable by array CGH. In this project, the genomic abnormalities were investigated in 32 MR patients using this technique.

MATERIALS AND METHODS:

Patients with IMR with dysmorphism were investigated in this study. Karyotype analysis, fragile X and metabolic tests were first carried out on the patients. The copy number variation was then assessed in a total of 32 patients with normal results for the mentioned tests using whole genome oligo array CGH. Multiple ligation probe amplification was carried out as a confirmation test.

RESULTS:

In total, 19% of the patients showed genomic abnormalities. This is reduced to 12.5% once the two patients with abnormal karyotypes (upon re-evaluation) are removed.

CONCLUSION:

The array CGH technique increased the detection rate of genomic imbalances in our patients by 12.5%. It is an accurate and reliable method for the determination of genomic imbalances in patients with IMR and dysmorphism.     

Comparative genomic assessment of Multi-Locus Sequence Typing: rapid accumulation of genomic heterogeneity among clonal isolates of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

Background  

Multi-Locus Sequence Typing (MLST) has emerged as a leading molecular typing method owing to its high ability to discriminate among bacterial isolates, the relative ease with which data acquisition and analysis can be standardized, and the high portability of the resulting sequence data. While MLST has been successfully applied to the study of the population structure for a number of different bacterial species, it has also provided compelling evidence for high rates of recombination in some species. We have analyzed a set of Campylobacter jejuni strains using MLST and Comparative Genomic Hybridization (CGH) on a full-genome microarray in order to determine whether recombination and high levels of genomic mosaicism adversely affect the inference of strain relationships based on the analysis of a restricted number of genetic loci.     

Comparative genomic hybridisation for the analysis of chromosomal imbalances in solid tumours and haematological malignancies

 Comparative genomic hybridisation (CGH) is based on a two-colour, competitive fluorescence in situ hybridisation of differentially labelled tumour and reference DNA to normal metaphase chromosomes. This new technology has made a great impact in molecular tumour pathology due to its possible application to archival specimens and the ability to create copy number karyotypes throughout the whole genome from very small amounts of DNA. If chromosomal imbalances can be correlated with a etiological and clinical features of tumours, CGH could be able to provide new prognostic and diagnostic criteria. CGH findings further provide starting points for the molecular genetic characterisation of altered chromosomal regions harbouring yet unidentified genes involved in tumorigenesis and tumour progression. An overview of the results of published CGH studies on solid tumours and haematological malignancies is presented. Methodological limitations of the CGH technology are reported, as well as future developments which will improve its use in routine analysis. Accepted: 29 July 1997     

A statistical approach for array CGH data analysis

Background  

Microarray-CGH experiments are used to detect and map chromosomal imbalances, by hybridizing targets of genomic DNA from a test and a reference sample to sequences immobilized on a slide. These probes are genomic DNA sequences (BACs) that are mapped on the genome. The signal has a spatial coherence that can be handled by specific statistical tools. Segmentation methods seem to be a natural framework for this purpose. A CGH profile can be viewed as a succession of segments that represent homogeneous regions in the genome whose BACs share the same relative copy number on average. We model a CGH profile by a random Gaussian process whose distribution parameters are affected by abrupt changes at unknown coordinates. Two major problems arise : to determine which parameters are affected by the abrupt changes (the mean and the variance, or the mean only), and the selection of the number of segments in the profile.     

An experimental loop design for the detection of constitutional chromosomal aberrations by array CGH

Background  

Comparative genomic hybridization microarrays for the detection of constitutional chromosomal aberrations is the application of microarray technology coming fastest into routine clinical application. Through genotype-phenotype association, it is also an important technique towards the discovery of disease causing genes and genomewide functional annotation in human. When using a two-channel microarray of genomic DNA probes for array CGH, the basic setup consists in hybridizing a patient against a normal reference sample. Two major disadvantages of this setup are (1) the use of half of the resources to measure a (little informative) reference sample and (2) the possibility that deviating signals are caused by benign copy number variation in the "normal" reference instead of a patient aberration. Instead, we apply an experimental loop design that compares three patients in three hybridizations.     

A unique combination of 17pter trisomy and 21qter monosomy in a boy with developmental delay,severe intellectual disability,growth retardation and dysmorphisms

Background

Microduplication at 17p13.3 and microdeletion at 21q22 are both rare chromosomal aberrations. The presence of both genomic imbalances in one patient has not been previously reported in literature. In this study, we performed a molecular diagnostic testing with a whole genome microarray on a 3-year-old boy with developmental delay, mental retardation and multiple malformations.

Methods

A routine G-banding karyotype analysis was performed using peripheral lymphocytes. Chromosome microarray analysis (CMA) was done using Affymetrix CytoScan™ HD array. Genomic imbalances were further confirmed by multiple ligation-dependent probe amplification (MLPA).

Results

The result of karyotyping was normal but CMA detected a 9.8 Mb microduplication at 17p13.3–13.1 (chr17: 1–9,875,545) and a 2.8 Mb microdeletion involving 21q22.3–qter (chr21: 45,239,077–48,097,372). The imbalances were due to a balanced translocation present in patient's mother. The patient was characterized with short stature, profound developmental delay, non-verbal, intellectual disability as well as craniofacial dysmorphism, subtle brain structural anomaly and sparse scalp hair.

Conclusions

This is the first patient reported with a combination of a microduplication at 17p13.3–13.1 and a microdeletion at 21q22.3–qter. Both genomic imbalances were undetected by conventional karyotyping but were delineated with CMA test. Synergistic effect from the two rare genomic imbalances is likely responsible for the severe clinical phenotypes observed in this patient.     

M-CGH: Analysing microarray-based CGH experiments

Background  

Microarray-based comparative genomic hybridisation (array CGH) is a technique by which variation in relative copy numbers between two genomes can be analysed by competitive hybridisation to DNA microarrays. This technology has most commonly been used to detect chromosomal amplifications and deletions in cancer. Dedicated tools are needed to analyse the results of such experiments, which include appropriate visualisation, and to take into consideration the physical relation in the genome between the probes on the array.     

Chromosome-breakage genomic instability and chromothripsis in breast cancer

Background

Chromosomal breakage followed by faulty DNA repair leads to gene amplifications and deletions in cancers. However, the mere assessment of the extent of genomic changes, amplifications and deletions may reduce the complexity of genomic data observed by array comparative genomic hybridization (array CGH). We present here a novel approach to array CGH data analysis, which focuses on putative breakpoints responsible for rearrangements within the genome.

Results

We performed array comparative genomic hybridization in 29 primary tumors from high risk patients with breast cancer. The specimens were flow sorted according to ploidy to increase tumor cell purity prior to array CGH. We describe the number of chromosomal breaks as well as the patterns of breaks on individual chromosomes in each tumor. There were differences in chromosomal breakage patterns between the 3 clinical subtypes of breast cancers, although the highest density of breaks occurred at chromosome 17 in all subtypes, suggesting a particular proclivity of this chromosome for breaks. We also observed chromothripsis affecting various chromosomes in 41% of high risk breast cancers.

Conclusions

Our results provide a new insight into the genomic complexity of breast cancer. Genomic instability dependent on chromosomal breakage events is not stochastic, targeting some chromosomes clearly more than others. We report a much higher percentage of chromothripsis than described previously in other cancers and this suggests that massive genomic rearrangements occurring in a single catastrophic event may shape many breast cancer genomes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-579) contains supplementary material, which is available to authorized users.     

Visualization-based discovery and analysis of genomic aberrations in microarray data

Background  

Chromosomal copy number changes (aneuploidies) play a key role in cancer progression and molecular evolution. These copy number changes can be studied using microarray-based comparative genomic hybridization (array CGH) or gene expression microarrays. However, accurate identification of amplified or deleted regions requires a combination of visual and computational analysis of these microarray data.     

Mitochondrial phylogenomics of the Bivalvia (Mollusca): searching for the origin and mitogenomic correlates of doubly uniparental inheritance of mtDNA

Background  

Doubly uniparental inheritance (DUI) is an atypical system of animal mtDNA inheritance found only in some bivalves. Under DUI, maternally (F genome) and paternally (M genome) transmitted mtDNAs yield two distinct gender-associated mtDNA lineages. The oldest distinct M and F genomes are found in freshwater mussels (order Unionoida). Comparative analyses of unionoid mitochondrial genomes and a robust phylogenetic framework are necessary to elucidate the origin, function and molecular evolutionary consequences of DUI. Herein, F and M genomes from three unionoid species, Venustaconcha ellipsiformis, Pyganodon grandis and Quadrula quadrula have been sequenced. Comparative genomic analyses were carried out on these six genomes along with two F and one M unionoid genomes from GenBank (F and M genomes of Inversidens japanensis and F genome of Lampsilis ornata).     

Performance of a 70-mer oligonucleotide microarray for genotyping of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

Background  

Campylobacter jejuni is widespread in the environment and is the major cause of bacterial gastroenteritis in humans. In the present study we use microarray-based comparative genomic hybridizations (CGH), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to analyze closely related C. jejuni isolates from chicken and human infection.     

Rearrangements of chromosome 18 illustrate the utility of array-based comparative genomic hybridization

Comprehensive and reliable testing is an important component of counseling and management in clinical genetics. Identification of imbalances of chromosomal segments has uncovered new genes and has established phenotype/genotype correlations for many syndromes with previously unidentified causes. Conventional cytogenetics has proven to be useful for the detection of large aberrations, but its resolution limits the identification of submicroscopic alterations. Comparative genomic hybridization (CGH) on a microarray-based platform has the potential to detect and characterize both microscopic and submicroscopic chromosomal abnormalities. Nine cases of aberrations involving chromosome 18 are used to illustrate the use and clinical potential of array CGH.     

SeeGH – A software tool for visualization of whole genome array comparative genomic hybridization data

Background  

Array comparative genomic hybridization (CGH) is a technique which detects copy number differences in DNA segments. Complete sequencing of the human genome and the development of an array representing a tiling set of tens of thousands of DNA segments spanning the entire human genome has made high resolution copy number analysis throughout the genome possible. Since array CGH provides signal ratio for each DNA segment, visualization would require the reassembly of individual data points into chromosome profiles.     

Analysis of the genome content of <Emphasis Type="Italic">Lactococcus garvieae</Emphasis> by genomic interspecies microarray hybridization

Background  

Lactococcus garvieae is a bacterial pathogen that affects different animal species in addition to humans. Despite the widespread distribution and emerging clinical significance of L. garvieae in both veterinary and human medicine, there is almost a complete lack of knowledge about the genetic content of this microorganism. In the present study, the genomic content of L. garvieae CECT 4531 was analysed using bioinformatics tools and microarray-based comparative genomic hybridization (CGH) experiments. Lactococcus lactis subsp. lactis IL1403 and Streptococcus pneumoniae TIGR4 were used as reference microorganisms.     

The core genome of the anaerobic oral pathogenic bacterium <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>

Background  

The Gram negative anaerobic bacterium Porphyromonas gingivalis has long been recognized as a causative agent of periodontitis. Periodontitis is a chronic infectious disease of the tooth supporting tissues eventually leading to tooth-loss. Capsular polysaccharide (CPS) of P. gingivalis has been shown to be an important virulence determinant. Seven capsular serotypes have been described. Here, we used micro-array based comparative genomic hybridization analysis (CGH) to analyze a representative of each of the capsular serotypes and a non-encapsulated strain against the highly virulent and sequenced W83 strain. We defined absent calls using Arabidopsis thaliana negative control probes, with the aim to distinguish between aberrations due to mutations and gene gain/loss.     

The first set of EST resource for gene discovery and marker development in pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis>L.)

Background  

Pigeonpea (Cajanus cajan (L.) Millsp) is one of the major grain legume crops of the tropics and subtropics, but biotic stresses [Fusarium wilt (FW), sterility mosaic disease (SMD), etc.] are serious challenges for sustainable crop production. Modern genomic tools such as molecular markers and candidate genes associated with resistance to these stresses offer the possibility of facilitating pigeonpea breeding for improving biotic stress resistance. Availability of limited genomic resources, however, is a serious bottleneck to undertake molecular breeding in pigeonpea to develop superior genotypes with enhanced resistance to above mentioned biotic stresses. With an objective of enhancing genomic resources in pigeonpea, this study reports generation and analysis of comprehensive resource of FW- and SMD- responsive expressed sequence tags (ESTs).