Molekularzytogenetische Methoden und Array-Diagnostik in der Pr?natalmedizin

In addition to the widely used cytogenetic standard approaches, molecular methods are being increasingly used in prenatal diagnostics. While molecular cytogenetics, e.g., fluorescence in situ hybridization (FISH), has been used for many years in invasive prenatal diagnostics, array-based diagnostics are only now being implemented in this field. FISH is prenatally applied for determination of size of a mosaic cell clone, for exclusion of a microdeletion, or for further clarification of structural chromosomal aberrations. Array CGH (comparative genomic hybridization) is used more conservatively in prenatal diagnostics, mostly for further clarification in sonographically abnormal fetuses and to diagnose breakpoints in cases with proven chromosomal changes. In the future, array CGH will gain further importance, but already provides a valuable supplement to the diagnostic approaches of the cytogenetic and the molecular-based methods.     

Clinical application of array-based comparative genomic hybridization for the identification of prognostically important genetic alterations in chronic lymphocytic leukemia

Genomic aberrations have increasingly gained attention as prognostic markers in B-cell chronic lymphocytic leukemia (CLL). Fluorescence in situ hybridization (FISH) has improved the detection rate of genomic alterations in CLL from approximately 50% using conventional cytogenetics to greater than 80%. More recently, array comparative genomic hybridization (CGH) has gained popularity as a clinical tool that can be applied to detect genomic gains and losses of prognostic importance in CLL. Array CGH and FISH are particularly useful in CLL because genomic gains and losses are key events with both biologic and prognostic significance, while balanced translocations have limited prognostic value. Although FISH has a higher technical sensitivity, it requires separate, targeted hybridizations for the detection of alterations at genomic loci of interest. Array CGH, on the other hand, has the ability to provide a genome-wide survey of genomic aberrations with a single hybridization reaction. Array CGH is expanding the known genomic regions of importance in CLL and allows these regions to be evaluated in the context of a genome-wide perspective. Ongoing clinical trials are evaluating the use of genomic aberrations as tools for risk-stratifying patients for therapy, thus increasing the need for reliable and high-yield methods to detect these genomic changes. In this review, we consider the use of array CGH as a clinical tool for the identification of genomic alterations with prognostic significance in CLL, and suggest ways to integrate this test into the clinical molecular diagnostic laboratory work flow.     

Development of bioinformatics resources for display and analysis of copy number and other structural variants in the human genome

The discovery of an abundance of copy number variants (CNVs; gains and losses of DNA sequences >1 kb) and other structural variants in the human genome is influencing the way research and diagnostic analyses are being designed and interpreted. As such, comprehensive databases with the most relevant information will be critical to fully understand the results and have impact in a diverse range of disciplines ranging from molecular biology to clinical genetics. Here, we describe the development of bioinformatics resources to facilitate these studies. The Database of Genomic Variants (http://projects.tcag.ca/variation/) is a comprehensive catalogue of structural variation in the human genome. The database currently contains 1,267 regions reported to contain copy number variation or inversions in apparently healthy human cases. We describe the current contents of the database and how it can serve as a resource for interpretation of array comparative genomic hybridization (array CGH) and other DNA copy imbalance data. We also present the structure of the database, which was built using a new data modeling methodology termed Cross-Referenced Tables (XRT). This is a generic and easy-to-use platform, which is strong in handling textual data and complex relationships. Web-based presentation tools have been built allowing publication of XRT data to the web immediately along with rapid sharing of files with other databases and genome browsers. We also describe a novel tool named eFISH (electronic fluorescence in situ hybridization) (http://projects.tcag.ca/efish/), a BLAST-based program that was developed to facilitate the choice of appropriate clones for FISH and CGH experiments, as well as interpretation of results in which genomic DNA probes are used in hybridization-based experiments.     

Integrated cytogenetic and high-resolution array CGH analysis of genomic alterations associated with MYCN amplification

Amplification of oncogenes and closely linked flanking genes is common in some types of cancer and can be associated with complex chromosome rearrangements and/or co-amplification of non-syntenic chromosomal regions. To better understand the etiology and structural complexity of focal MYCN amplicons in human neuronal cancer, we investigated the precise chromosomal locations of high copy number genomic regions in MYCN amplified cell lines. An integrated cytogenetic map of the MYCN amplicon was created using high-resolution array CGH, spectral karyotyping (SKY), multi-color banding (mBAND), and fluorescence in situ hybridization (FISH) in 4 human neuronal tumor cell lines. The evidence of complex intra- and inter-chromosomal events, providing clues concerning the nature of the genomic mechanisms that contributed to the process of MYCN amplification, was observed. The presence of multiple co-amplified syntenic or non-syntenic sequences in the MYCN amplicon is quite intriguing. MYCN is usually centrally located in the amplicon; however, the structure and complexity of the amplicons were highly variable. It is noteworthy that clusters of unstable repetitive regions characterized by CNV sequences were present throughout the regions encompassed by MYCN gene amplification, and these sequences could provide a mechanism to destabilize this region of the genome. Complex structural rearrangements involving genomic losses and gains in the 2p24 region lead to MYCN amplification and that these rearrangements can trigger amplification events.     

Single-cell isolation from cell suspensions and whole genome amplification from single cells to provide templates for CGH analysis

A comprehensive genomic analysis of single cells is instrumental for numerous applications in tumor genetics, clinical diagnostics and forensic analyses. Here, we provide a protocol for single-cell isolation and whole genome amplification, which includes the following stages: preparation of single-cell suspensions from blood or bone marrow samples and cancer cell lines; their characterization on the basis of morphology, interphase fluorescent in situ hybridization pattern and antibody staining; isolation of single cells by either laser microdissection or micromanipulation; and unbiased amplification of single-cell genomes by either linker-adaptor PCR or GenomePlex library technology. This protocol provides a suitable template to screen for chromosomal copy number changes by conventional comparative genomic hybridization (CGH) or array CGH. Expected results include the generation of several micrograms of DNA from single cells, which can be used for CGH or other analyses, such as sequencing. Using linker-adaptor PCR or GenomePlex library technology, the protocol takes 72 or 30 h, respectively.     

The genetic screening of preimplantation embryos by comparative genomic hybridisation

Comparative genomic hybridization (CGH) is an indirect DNA-based test which allows for the accurate analysis of aneuploidy involving any of the 24 types of chromosomes present (22 autosomes and the X and Y sex chromosomes). Traditionally, embryos have been screened using fluorescence in situ hybridization (FISH)--a technique that was limited in the number of chromosomes able to be identified in any one sample. Early CGH reports on aneuploidy in preimplantation embryos showed that any of the 24 chromosomes could be involved and so FISH methods were going to be ineffective in screening out abnormal embryos. Our results from routine clinical application of array CGH in preimplantation genetic diagnosis (PGD) patients confirm previous reports on patterns of chromosomal contribution to aneuploidy. The pregnancy outcomes following embryo transfer also indicate that despite the requirement to freeze embryos, rates are encouraging, and successful ongoing pregnancies can be achieved.     

Proximal 19q trisomy: a new syndrome of morbid obesity and mental retardation

AIMS: To report on the clinical and metabolic characteristic and the unique chromosomal defect of a mentally retarded and morbidly obese patient. METHODS: A 13-year follow-up, including insulin sensitivity, lipid profile and polysomnography studies and various therapeutic interventions are described. The presence of a supernumerary marker in karyotype preparation was further studied by fluorescence in situ hybridization (FISH). Comparative genomic hybridization (CGH) was used to identify the source of the chromosomal marker. RESULTS: Insulin resistance was found by the homeostatic model assessment (HOMA) and the quantitative insulin sensitivity check index (QUICKI). M-FISH identified euchromatin derived from chromosome 19, and CGH confirmed the FISH results and demonstrated that the supernumerary marker derived from 19q12 to 19q13.2. CONCLUSION: The clinical and metabolic characteristics in association with partial chromosomal trisomy differ our patient from the currently known syndromes of obesity and mental retardation. The metabolic impairments in this case can derive from unbalanced expression of several genes in the 19q12-19q13.2 region, genes that are related to adipose tissue homeostasis and insulin resistance. The clinical and genetic similarities to a previously reported case may suggest that partial 19q trisomy is a new syndrome of obesity and mental retardation.     

Structural unbalanced chromosome rearrangements resolved by comparative genomic hybridization.

The identification of unbalanced structural chromosome rearrangements using conventional cytogenetic techniques depends on recognition of the unknown material from its banding pattern. Even with optimally banded chromosomes, when large chromosome segments are involved, cytogeneticists may not always be able to determine the origin of extrachromosomal material and supernumerary chromosomes. We report here on the application of comparative genomic hybridization (CGH), a new molecular-cytogenetic assay capable of detecting chromosomal gains and losses, to six clinical samples suspected of harboring unbalanced structural chromosome abnormalities. CGH provided essential information on the nature of the unbalanced aberration investigated in five of the six samples. This approach has proved its ability to resolve complex karyotypes and to provide information when metaphase chromosomes are not available. In cases where metaphase chromosome spreads were available, confirmation of CGH results was easily obtained by fluorescence in situ hybridization (FISH) using specific probes. Thus the combined use of CGH and FISH provided an efficient method for resolving the origin of aberrant chromosomal material unidentified by conventional cytogenetic analysis.     

Uses and limitations of two molecular cytogenetic techniques for the study of arrested embryos obtained through assisted reproduction technology

We studied chromosomal abnormalities in arrested embryos produced by assisted reproductive technology with fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) in order to determine the best technique for evaluating chromosomal aneusomies to be implemented in different situations. We examined individual blastomeres from arrested embryos by FISH and arrested whole embryos by CGH. All of the 10 FISH-analyzed embryos gave results, while only 7 of the 30 embryos analyzed by CGH were usable. Fifteen of the 17 embryos were chromosomally abnormal. CGH provided more accurate data for arrested embryos; however, FISH is the technique of choice for screening in preimplantation genetic diagnosis, because the results can be obtained within a day, while the embryos are still in culture.     

Genetics of early miscarriage

A miscarriage is the most frequent complication of a pregnancy. Poor chromosome preparations, culture failure, or maternal cell contamination may hamper conventional karyotyping. Techniques such as chromosomal comparative genomic hybridization (chromosomal‐CGH), array-comparative genomic hybridization (array-CGH), fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA) and quantitative fluorescent polymerase chain reaction (QF-PCR) enable us to trace submicroscopic abnormalities. We found the prevalence of chromosome abnormalities in women facing a single sporadic miscarriage to be 45% (95% CI: 38–52; 13 studies, 7012 samples). The prevalence of chromosome abnormalities in women experiencing a subsequent miscarriage after preceding recurrent miscarriage proved to be comparable: 39% (95% CI: 29–50; 6 studies 1359 samples). More chromosome abnormalities are detected by conventional karyotyping compared to FISH or MLPA only (chromosome region specific techniques), and the same amount of abnormalities compared to QF-PCR (chromosome region specific techniques) and chromosomal‐CGH and array-CGH (whole genome techniques) only. Molecular techniques could play a role as an additional technique when culture failure or maternal contamination occurs: recent studies show that by using array-CGH, an additional 5% of submicroscopic chromosome variants can be detected. Because of the small sample size as well as the unknown clinical relevance of these molecular aberrations, more and larger studies should be performed of submicroscopic chromosome abnormalities among sporadic miscarriage samples. For recurrent miscarriage samples molecular technique studies are relatively new. It has often been suggested that miscarriages are due to chromosomal abnormalities in more than 50%, but the present review has determined that chromosomal and submicroscopic genetic abnormalities on average are prevalent in maximally half of the miscarriage samples. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.     

A novel 5q11.2 deletion detected by microarray comparative genomic hybridisation in a child referred as a case of suspected 22q11 deletion syndrome

The 22q11 deletion syndrome (22q11DS) is a developmental syndrome comprising of heart, palate, thymus and parathyroid glands defects. Individuals with 22q11DS usually carry a 1.5- to 3-Mb heterozygous deletion on chromosome 22q11.2. However, there are many patients with features of 22q11DS without a known cause from conventional karyotype and FISH analysis. Six patients with features of 22q11DS, a normal chromosomal and FISH 22q11 analysis, were selected for investigation by microarray genomic comparative hybridisation (array CGH). Array-CGH is a powerful technology enabling detection of submicroscopic chromosome duplications and deletions by comparing a differentially labelled test sample to a control. The samples are co-hybridised to a microarray containing genomic clones and the resulting ratio of fluorescence intensities on each array element is proportional to the DNA copy number difference. No chromosomal changes were detected by hybridisation to a high resolution array representing chromosome 22q. However, one patient was found to have a 6-Mb deletion on 5q11.2 detected by a whole genome 1-Mb array. This deletion was confirmed with fluorescence in-situ hybridisation (FISH) and microsatellite marker analysis. It is the first deletion described in this region. The patient had tetralogy of Fallot, a bifid uvula and velopharyngeal insufficiency, short stature, learning and behavioural difficulties. This case shows the increased sensitivity of array CGH over detailed karyotype analysis for detection of chromosomal changes. It is anticipated that array CGH will improve the clinicians capacity to diagnose congenital syndromes with an unknown aetiology.     

Comparative genomic hybridization in combination with flow cytometry improves results of cytogenetic analysis of spontaneous abortions

More than 50% of spontaneous abortions (SAs) have abnormal chromosomes; the most common abnormalities are trisomy, sex chromosome monosomy, and polyploidy. Conventional cytogenetic analysis of SAs depends on tissue culturing and is associated with a significant tissue culture failure rate and contamination by maternally derived cells. Comparative genomic hybridization (CGH), in combination with flow cytometry (FCM), can detect numerical and unbalanced structural chromosomal abnormalities associated with SAs while avoiding the technical problems associated with tissue culture. Routine cytogenetic and CGH analysis was performed independently on tissue from 301 SAs. Samples shown to be chromosomally balanced by CGH were analyzed by FCM to determine ploidy. Of 253 samples successfully analyzed by both approaches, there was an absolute correlation of results in 235 (92.8%). Of the 18 cases with discrepancies between cytogenetic and CGH/FCM results, an explanation could be found in 17. Twelve samples produced a 46,XX karyotype by cytogenetics, whereas CGH/FCM demonstrated aneuploidy/polyploidy or a male genome, indicating maternal contamination of the tissue cultures. In two cases, where tetraploidy was demonstrated by cytogenetics and diploidy by FCM, tissue culture artifact is implied. In three cases, CGH demonstrated an aneuploidy, and cytogenetics demonstrated hypertriploidy. In one unexplainable case, aneuploidy demonstrated by CGH could not be detected by repeat CGH analysis, conventional cytogenetic, or FISH analysis. These results demonstrate that CGH supplemented with FCM can readily identify chromosomal abnormalities associated with SAs and, by avoiding maternal contamination and tissue culture artifacts, can do so with a lower failure rate and more accuracy than conventional cytogenetic analysis.     

Diagnostic genome profiling in mental retardation

Mental retardation (MR) occurs in 2%-3% of the general population. Conventional karyotyping has a resolution of 5-10 million bases and detects chromosomal alterations in approximately 5% of individuals with unexplained MR. The frequency of smaller submicroscopic chromosomal alterations in these patients is unknown. Novel molecular karyotyping methods, such as array-based comparative genomic hybridization (array CGH), can detect submicroscopic chromosome alterations at a resolution of 100 kb. In this study, 100 patients with unexplained MR were analyzed using array CGH for DNA copy-number changes by use of a novel tiling-resolution genomewide microarray containing 32,447 bacterial artificial clones. Alterations were validated by fluorescence in situ hybridization and/or multiplex ligation-dependent probe amplification, and parents were tested to determine de novo occurrence. Reproducible DNA copy-number changes were present in 97% of patients. The majority of these alterations were inherited from phenotypically normal parents, which reflects normal large-scale copy-number variation. In 10% of the patients, de novo alterations considered to be clinically relevant were found: seven deletions and three duplications. These alterations varied in size from 540 kb to 12 Mb and were scattered throughout the genome. Our results indicate that the diagnostic yield of this approach in the general population of patients with MR is at least twice as high as that of standard GTG-banded karyotyping.     

Molecular cytogenetics in metaphase and interphase cells for cancer and genetic research, diagnosis and prognosis. Application in tissue sections and cell suspensions

As the pioneer among molecular cytogenetics techniques, fluorescence in situ hybridization (FISH) allows identification of specific sequences in a structurally preserved cell, in metaphase or interphase. This technique, based on the complementary double-stranded nature of DNA, hybridizes labeled specific DNA (probe). The probe, bound to the target, will be developed into a fluorescent signal. The fact that the signal can be detected clearly, even when fixed in interphase, improves the accuracy of the results, since in some cases it is extremely difficult to obtain mitotic samples. FISH is still used mostly in research, but there are diagnostic applications. New nomenclature is being developed in order to define many of the aberrations that were not distinguished before FISH. Prenatal diagnosis of aneuploidies and malignancies are promptly detected with FISH, which is very useful in critical cases. In some tumors, where chromosomal abnormalities are too complicated to classify manually, the technique of comparative genomic hybridization (CGH), a competitive FISH, allows examiners to determine complete or partial gain or loss of chromosomes. CGH results allow the classification of many tumor cell lines and along with other complementary techniques, like microdissection-FISH, PRINS, etc., increase the possibility of choosing an appropriate treatment for cancer patients.     

From microscopes to microarrays: dissecting recurrent chromosomal rearrangements

Submicroscopic chromosomal rearrangements that lead to copy-number changes have been shown to underlie distinctive and recognizable clinical phenotypes. The sensitivity to detect copy-number variation has escalated with the advent of array comparative genomic hybridization (CGH), including BAC and oligonucleotide-based platforms. Coupled with improved assemblies and annotation of genome sequence data, these technologies are facilitating the identification of new syndromes that are associated with submicroscopic genomic changes. Their characterization reveals the role of genome architecture in the aetiology of many clinical disorders. We review a group of genomic disorders that are mediated by segmental duplications, emphasizing the impact that high-throughput detection methods and the availability of the human genome sequence have had on their dissection and diagnosis.     

Single-cell copy number variation detection

Detection of chromosomal aberrations from a single cell by array comparative genomic hybridization (single-cell array CGH), instead of from a population of cells, is an emerging technique. However, such detection is challenging because of the genome artifacts and the DNA amplification process inherent to the single cell approach. Current normalization algorithms result in inaccurate aberration detection for single-cell data. We propose a normalization method based on channel, genome composition and recurrent genome artifact corrections. We demonstrate that the proposed channel clone normalization significantly improves the copy number variation detection in both simulated and real single-cell array CGH data.     

Exon array CGH: detection of copy-number changes at the resolution of individual exons in the human genome

The development of high-throughput screening methods such as array-based comparative genome hybridization (array CGH) allows screening of the human genome for copy-number changes. Current array CGH strategies have limits of resolution that make detection of small (less than a few tens of kilobases) gains or losses of genomic DNA difficult to identify. We report here a significant improvement in the resolution of array CGH, with the development of an array platform that utilizes single-stranded DNA array elements to accurately measure copy-number changes of individual exons in the human genome. Using this technology, we screened 31 patient samples across an array containing a total of 162 exons for five disease genes and detected copy-number changes, ranging from whole-gene deletions and duplications to single-exon deletions and duplications, in 100% of the cases. Our data demonstrate that it is possible to screen the human genome for copy-number changes with array CGH at a resolution that is 2 orders of magnitude higher than that previously reported.