Modern molecular cytogenetic techniques in genetic diagnostics

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1 246 Research Update Techniques & Applications Modern molecular cytogenetic techniques in genetic diagnostics Holger Tönnies During the past decade, fluorescence in situ hybridization (FISH) has become an important complementing application in genetic diagnostics. The use of variable FISH techniques enhances the thorough interpretation of numerical and complex chromosome aberrations, bridging the gap between conventional chromosome banding analysis and molecular genetic studies. This review gives a brief overview of the different molecular cytogenetic FISH techniques and applications currently used in routine genetic diagnostics and focus on their advantages and limitations. Published online: 8 May 2002 Conventional cytogenetic karyotype analysis is still the standard test in genetic diagnostics to identify numerical and structural chromosomal aberrations, which are the major cause of mental and psychomotoric retardation, miscarriages, congenital anomalies, and also common findings in neoplasia. Aberrant cytogenetic findings relevant for diagnosis, clinical management, and appropriate genetic counseling have contributed enormously to the definition of phenotype karyotype correlations in chromosomal syndromes and hematological diseases. However, the limited chromosome specific banding resolution obtained by Giemsa banding (GTG-banding) makes the recognition and interpretation of masked or cryptic chromosome aberrations difficult to ascertain and is therefore potentially inaccurate. We want to see more in situ hybridization In the late 1960s, Pardue and Gall [1] first described the in situ hybridization (ISH) of radioactively labeled repetitive probes on cytological preparations. In 1986, Pinkel et al. [2] and Cremer et al. [3] reported fluorescence in situ hybridization (FISH) using non-radioactively labeled probes. FISH is based on the hybridization of complementary, single-stranded nucleic acids to fixed target genetic material (i.e. metaphase chromosomes or interphase nuclei). FISH probes are labeled either directly using fluorochrome-conjugated nucleotides (e.g. fluorescein-dutp) or indirectly using reporter molecules (e.g. biotin-dutp) by nick-translation, random priming, PCR, or various other molecular genetic techniques. By this sensitive method, nucleic acid probes allow the exploration of the presence, number and distinct location (in situ) of genetic material by forming a new duplex with the complementary target material. Fluorescent signals of bound probes are inspected using a filter-equipped epifluorescence microscope and computer software. Which probe for which problem? A large number of different probes designed to identify specific chromosomes and parts thereof are available for diagnostic purposes (Table 1). The choice of probe and the simultaneous use of multi-probe essays depends on the particular application in question. For chromosome enumeration, probes detecting chromosome-specific α-satellite regions near the centromeres are preferred [centromeric probe (cep)] (Fig 1b). For interphase cell analysis, which can be performed in cytological preparations as well as in sections of formaldehyde-fixed and paraffin-embedded tissues, these probes are particularly attractive because analysis is rapid and sensitivity is high. However, by using these probes for interphase cytogenetics, centromeric polymorphisms can result in false positive or false negative results. Recently, an all-human centromere-specific multicolour-fish approach (cenm-fish) for the ascertainment of the origin of structurally abnormal, cytogenetically unidentifiable chromosomes so-called marker chromosomes was reported by Nietzel et al. [4]. This technique allows the simultaneous characterization of all human centromeres using differently labeled centromeric satellite as probes. However, by using cep probes, only the centromeric parts of chromosomes can be explored, so that other chromosome abnormalities affecting the euchromatin, are excluded from investigation. Chromosome painting probes [whole chromosome paint (wcp) and partial chromosome paint (pcp)] obtained by flow sorting or micromanipulator-mediated chromosome microdissection, allow the labeling of individual chromosomes in metaphase spreads and the identification of both numerical and interchromosomal structural rearrangements (Fig. 1a). The use of chromosome paints to ascertain chromosomal aberrations needs prior knowledge of the affected chromosome(s) in question. However, a locus-specific determination of the affected chromosomal region in intrachromosomal rearrangements such as deletions, duplications and paracentric inversions, and the detection of interchromosomal translocations involving only small regions of the chromosome ends (less well represented in these probes), cannot be performed successfully by using these probes. Appropriate region-specific cloned probes [e.g. yeast artificial chromosome (YACs), bacterial artificial chromosome (BACs) and P1 artificial chromosome (PACs)] are used for the determination and characterization of intra- and interchromosomal rearrangements regardless of their complexity (Fig. 1c). For the investigation of specific submicroscopic chromosomal regions, a wide spectrum of so-called locus-specific identifiers (LSI) are commercially available for the detection of the classical microdeletion syndromes (contiguous gene syndromes) or specific chromosome translocations followed by chimeric gene fusion common in hematological diseases (e.g. BCR/ABL). Special subtelomeric probes were established to scan metaphase spreads for cryptic aberrations at the gene-rich ends of the chromosomes, probably accounting for 5 10% of unexplained moderate-to-severe mental retardation cases [5]. Recently, /02/$ see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S (02)

2 Research Update 247 Table 1. Molecular cytogenetic techniques used in genetic diagnostics Technique Probe used Hybridization target Diagnostic applications Advantages Limitations FISH Cep a Specimen Chromosome enumeration, marker Simple setup and rapid results a, Small spectrum of analysis a, metaphase chromosome identification no need for specific cytogenetic no information about spreads or expertise, applicable on archived euchromatic content in interphase material marker chromosomes nuclei Wcp and pcp Specimen Interchromosomal aberrations Simple setup and fast detection of Small spectrum of analysis, need metaphase (translocations) and chromosome interchromosomal aberrations in for prior knowledge of affected spreads enumeration question chromosome, locus-specific determination of intrachromosomal aberrations not possible, need for proliferating material, subtelomeric regions underrepresented Cloned probes Specimen Inter- and intrachromosomal High sensitivity and specificity, Small spectrum of analysis, need as metaphase aberrations [translocations, detection of gene amplification of prior knowledge of affected YAC/BAC/PAC spreads or inversions, microdeletions and possible (e.g. oncogenes) chromosome locus or genes etc. b interphase microduplications, chimeric gene nuclei fusions (leukemias)] Subtelomeric Specimen Cryptic subtelomeric High sensitivity and specificity Small euchromatic spectrum of probes c metaphase rearrangements analysis, proliferating material spreads Micro-FISH Microdissected Specimen and Marker chromosome Straightforward and complete Technically demanding, need for chromosomes control characterization by forward euchromatic characterization of excellent cytogenetic expertise or parts thereof metaphase (specimen) and reverse (control) chromosome in question, for chromosome dissection spreads painting especially of marker chromosomes (mosaics) Chromosomal YAC clones, Specimen Inter- and intrachromosomal Metaphase wide detection of Poor resolution, insensitive locusbar codes fragment metaphase aberrations gross chromosome alterations specific determination of hybrids spreads interchromosomal aberrations and break-point detection, staining gaps Rx-FISH Cross-species Specimen Inter- and intrachromosomal Metaphase wide detection of Poor resolution, insensitive locus wcp-mix metaphase aberrations gross chromosome alterations specific determination of spreads interchromosomal aberrations and break-point detection Multicolor- Region-specific Specimen Intrachromosomal aberrations Sensitive detection of inversion Technically demanding, costs per banding probes of one metaphase breakpoints, locus specific chromosome, information only (MCB) chromosome spreads information (chromosomal about the chromosome probes band), high reproducibility hybridized SKY/M-FISH All human Specimen Interchromosomal aberrations as Whole-metaphase scanning, no Need for proliferating material, wcp-mix metaphase translocations, chromosome need for prior knowledge of insensitive locus-specific spreads enumeration, marker chromosome chromosomes affected, sensitive determination of identification translocation and marker intrachromosomal aberrations chromosome identification and break-point detection, costs, technically demanding CGH Specimen Control Chromosomal and genetic inter-, Whole genome scanning technique, No detection of balanced metaphase and intrachromosomal imbalances no need of proliferating material, aberrations or small imbalances, spreads gene amplifications, marker applicable on archived material, need of good cytogenetic chromosome characterization detection of gene amplification, expertise for karyotyping, missing locus-specific information of of small mosaics imbalances (chromosomal band) Matrix-CGH Specimen Defined Chromosomal and genetic inter- Scanning of a large informative No detection of balanced probes and intrachromosomal probe-set, individual array aberrations or small imbalances imbalances, gene amplifications, construction, no need for not displayed by the probe set, marker chromosome proliferating material, sensitive missing of small mosaics, costs, characterization detection of imbalances, no technically demanding cytogenetic expertise, automated signal interpretation Abbreviations: BAC, bacterial artificial chromosome; cep, centromeric probe; cenm-fish, centromere-specific multicolour-fish; CGH, comparative genomic hybridization; FISH, fluorescent in situ hybridization; LSI, locus-specific identifiers; M-FISH, multicolor FISH; PAC, P1 artificial chromosome; pcp, partial chromosome paint; Rx, cross species; SKY, spectral karyotyping; TM-FISH, telomeric multicolour FISH; wcp, whole chromosome paint; YAC, yeast artificial chromosome. a including cenm-fish b including commercially available LSI probes like bcr abl and microdeletion probes. c including TM-FISH

3 248 Research Update Henegariu et al. [6] reported the analysis of 41 chromosome ends simultaneously using a multicolour hybridization assay including probes located near the end of these chromosomes (0.1 1 Mb from the telomere) (TM-FISH). FISH probes: make your own one In addition to the use of specific commercial or non-commercial cloned probes, micro-manipulated chromosome microdissection, followed by PCR-mediated amplification, labeling and reverse (on normal metaphase spread) or forward (on affected cell) painting is a straightforward, highly informative strategy to characterize marker and other aberrant chromosomes (micro-fish [7]). However, there are practical limitations to micro-fish for diagnostic purposes as specialized equipment, technical skill, and a profound cytogenetic knowledge are necessary. Colourful chromosome world The age of multicolour FISH(M-FISH)- based chromosome banding techniques started with chromosomal bar codes [8,9] and cross-species chromosome painting (Rx-FISH [10]) covering all human chromosomes in one experiment providing new possibilities for the identification of gross intra- and interchromosomal aberrations. However, the spatial resolution of ~100 bands per haploid chromosome set is low in comparison with standard GTG-banding (~400 bands) and makes these techniques less useful for primary diagnostic screening purposes. Another recently developed single chromosome multicolour-banding (MCB) technique allows a higher resolution independent of chromosome condensation [11,12]. This technique, based on the hybridization of differently labeled overlapping microdissection probes followed by computer-based assignment of distinctive pseudo-colours, can be preferentially used for the detection of intrachromosomal aberrations as inversions and deletions. To date, this technique is limited to a single chromosome pair at a time and thus needs the prior knowledge of the chromosome affected. Metaphase and genome scanning techniques Today, among the various locus- and chromosome-specific assays described here, which often require the hybridization of a whole repertoire of different probes to (a) (b) (c) TRENDS in Molecular Medicine Fig. 1. Fluorescence in situ hybridization (FISH) applications. (a) Metaphase spread hybridized with whole chromosome painting probes for chromosome 10 (red) and 17 (green) detecting a whole arm translocation (arrows). (b) Metaphase spread of the same patient detecting the centromere origin of the two derivative chromosomes (arrows) by hybridization of centromere probes for chromosome 10 (green) and 17 (red). (c) Hybridization of locus-specific probes for chromosome 15 (green: centromere; red proximal 15q: critical region; red distal 15q: control region; [VYSIS]) detecting a deletion of both the centromere region and the Prader Willi/Angelman critical region in a derivative chromosome 15 as a result of an unbalanced translocation (arrow). narrow down the overall composition of a chromosome aberration, some unbiased whole-metaphase or genome scanning approaches are established for the characterization of highly rearranged and unbalanced chromosome aberrations. Spectral karyotyping (SKY [13]) using interferometer-based spectral imaging and multicolour-fish [14] using different fluorochrome-specific filter sets are based on the simultaneously hybridization of 24 differently labeled chromosome painting probes on non-overlapping target metaphase spreads of the specimen. By the use of computer-generated false-colour chromosome images, non-homologous structural and numerical chromosomal aberrations can be easily detected in single metaphase spreads. The main advantage of both techniques is that no prior knowledge of affected chromosome(s) is required. These techniques are capable of detecting chromosome rearrangements such as translocations and euchromatic marker chromosomes in a single hybridization, even if the chromosome morphology is poor as in solid tumours. The major drawback for routine use is the poor sensitivity to detect aberrations like intrachromosomal deletions, duplications and inversions. The large number of probes, labeled with different fluorochromes, and the special technical equipment required, in particular for SKY analyses, restrict these scanning techniques to some specialized laboratories. Cytogenetics on : how does that work? Comparative genomic hybridization (CGH) [15] is a scanning technique based on the use of genomic as a probe (Fig. 2). CGH is a potent and reliable hybridization approach, which allows the comprehensive analysis of the entire genome in just one experiment providing information not only about the size of all chromosomal imbalances detected but also about their chromosomal band-specific assignment. CGH is based on the cohybridization of labeled whole genomic test and control in a ratio of 1:1 on normal control metaphase spreads under conditions of in situ suppression hybridization (CISS). After image capturing and karyotyping, quantification of fluorescence intensities over the entire length of each chromosome is performed by computer software calculating a copy-number karyotype reflecting chromosomal imbalances (Fig. 2). In contrast to other primary scanning techniques such as M-FISH and SKY, no proliferating material of the specimen is necessary for screening. This allows to study any specimen for which is available (e.g. paraffin-embedded tissue sections) for chromosomal imbalances on a band-specific cytogenetic level (~10 Mb). For example, CGH-based investigation of supernumerary euchromatic marker

4 Research Update 249 chromosomes provides information, not only on the chromosomal assignment but also on the chromosomal band-specific origin. Nevertheless, CGH cannot reveal balanced chromosomal rearrangements, often found in routine tumour genetics. Furthermore, by using whole genomic, CGH is not sufficiently sensitive to detect aberrations occurring in a small amount of cells reflecting a cell-to-cell heterogeneity (mosaic), a characteristic feature of carcinomas and leukemias. Nevertheless, these FISH-based scanning techniques, nowadays also utilized in pre- and postnatal diagnostics, are excellent pre-informative tools for the detection of chromosomal alterations, later to be further characterized by more sensitive, complementary methods using locus-specific probes [16 18]. Test (a) Cot-1 Control Hybridization CGH Karyotyping Image processing Microscopy Normal metaphase chromosomes DIM ENH Copy-number karyotype DIM Future developments Recent advances, such as improved hybridization protocols and strategies, novel commercial and non-commercial probe sets, alongside the efforts of the Human Genome Project, coupled with hardware development such as image analysis software and specific filter sets for newly established fluorochromes, facilitate the collection of new important data for the understanding of genetic diseases. Other current research-based applications, too numerous be listed in this paper, are likely to translate onto the growing list of clinically useful applications in the near future (for overview see Ref. [19]). For diagnostic purposes, new time- and cost-saving approaches with the potential to investigate high numbers of genetic loci in an automated fashion and with high resolution independently of cell culture and chromosome preparations are favoured. First steps in this direction are now being taken. In 1997, Solinas-Toldo and co-workers established a matrix-based CGH array (Matrix-CGH [20]) that replaces condensed metaphase chromosomes by well-defined cloned probes immobilized on a glass surface as hybridization target, allowing automated analysis of chromosomal imbalances such as microdeletions and overrepresentations. In 1998, Pinkel et al. [21] obtained new information on chromosomal imbalances in breast cancer using a chromosome 20-specific CGH array, and one year later, Pollack et al. [22] reported the successful application of c as target in (b) Matrix-CGH Spotted s their arrays for the mapping of copy-number changes. Recently, Snijders et al. [23] established an array using 2400 representations of BAC s across the human genome to detect imbalances in diploid, polyploid and heterogeneous backgrounds. Today, the probability of providing useful extra diagnostic information for genetic counseling, prognostics, and disease monitoring depends on the distinct molecular cytogenetic technique, probe selection and hybridization target material available. The nearer future will include specific diagnostic probe sets detecting cryptic subtelomeric chromosome aberrations and microdeletions in clinical genetics and specific chromosomal translocations in leukemias and lymphomas by simple, time-saving devices. Nevertheless, array techniques will not be capable of detecting low-level mosaicism and balanced rearrangements. At least for the time being, array screening has to be seen as a supplementing technique for the examination of chromosome aberrations in genetic diagnostics. Scanning CGH array ENH TRENDS in Molecular Medicine Fig. 2. Comparative genomic hybridization (CGH) and matrix-comparative genomic hybridization procedures. (a) Differentially labeled s are hybridized on control metaphase spreads. Copy-number aberrations are detected by measuring fluorescence intensity differences depicted in a copy-number karyotype. (b) Labeled s are hybridized on defined spotted clones reflecting the human genome or parts thereof. The resulting relative fluorescence intensities are measured by computer scanning. Abbreviations: Cot-1, highly repetitive human ; DIM, diminished (deletion); ENH, enhanced (duplication/amplification). Acknowledgements I am grateful to Heidemarie Neitzel for critical reading of the manuscript and to Antje Gerlach and Britta Teubner for their excellent technical assistance. References 1 Pardue, M.L. and Gall, J.G. (1969) Molecular hybridization of radioactive to the of cytological preparations. Proc. Natl. Acad. Sci. U. S. A. 64, Pinkel, D. et al. (1986) Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. U. S. A. 83, Cremer, T. et al. (1986) Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target s with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84. Hum. Genet. 74, Nietzel, A. et al. (2001) A new multicolor-fish approach for the characterization of marker chromosomes: centromere-specific multicolor-fish (cenm-fish). Hum. Genet. 108, Knight, S.J. et al. (1997) Development and clinical application of an innovative fluorescence in situ hybridization technique which detects submicroscopic rearrangements involving telomeres. Eur. J. Hum. Genet. 5, Henegariu, O. et al. (2001) Cryptic translocation identification in human and mouse using several telomeric multiplex fish (TM-FISH) strategies. Lab. Invest. 81,

5 250 Research Update 7 Meltzer, P.S. et al. (1992) Rapid generation of region specific probes by chromosome microdissection and their application. Nat. Genet. 1, Lengauer, C. et al. (1993) Chromosomal bar codes produced by multicolor fluorescence in situ hybridization with multiple YAC clones and whole chromosome painting probes. Hum. Mol. Genet. 2, Müller, S. et al. (1997) Toward a multicolor chromosome bar code for the entire human karyotype by fluorescence in situ hybridization. Hum. Genet. 100, Müller, S. et al. (1998) Cross-species colour segmenting: a novel tool in human karyotype analysis. Cytometry 33, Chudoba, I. et al. (1999) High resolution multicolor-banding: a new technique for refined FISH analysis of human chromosomes. Cytogenet. Cell Genet. 84, Mrasek, K. et al. (2001) Reconstruction of the female Gorilla gorilla karyotype using 25-color FISH and multicolor banding (MCB). Cytogenet. Cytogenet. Cell Genet. 93, Schröck, E. et al. (1996) Multicolor spectral karyotyping of human chromosomes. Science 273, Speicher, M.R. et al. (1996) Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat. Genet. 12, Kallioniemi, A. et al. (1992) Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258, Schröck, E. et al. (1997) Spectral karyotyping refines cytogenetic diagnostics of constitutional chromosomal abnormalities. Hum. Genet. 101, Uhrig, S. et al. (1999) Multiplex-FISH for pre- and postnatal diagnostic applications. Am. J. Hum. Genet. 65, Tönnies, H. et al. (2001) Comparative genomic based strategy for the analysis of different chromosome imbalances detected in conventional cytogenetic diagnostics. Cytogenet. Cell Genet. 93, Schwarzacher and Heslop-Harrison (2000) Practical in situ Hybridization, BIOS Scientific Publishers Limited 20 Solinas-Toldo, S. et al. (1997) Matrix-based comparative genomic hybridization: biochips to screen for genomic imbalances. Genes Chromosomes Cancer 20, Pinkel, D. et al. (1998) High resolution analysis of copy number variation using comparative genomic hybridization to microarrays. Nat. Genet. 20, Pollack, J.R. et al. (1999) Genome-wide analysis of copy-number changes using c microarrays. Nat. Genet. 23, Snijders, A.M. et al. (2001) Assembly of microarrays for genome-wide measurement of copy number. Nat. Genet. 29, Holger Tönnies Charité, Campus-Virchow, Institute of Human Genetics, Humboldt-University Berlin, Augustenburger Platz 1, D Berlin, Germany. Holger.Toennies@charite.de High-throughput protein arrays: prospects for molecular diagnostics Gerald Walter, Konrad Büssow, Angelika Lueking and Jörn Glökler High-throughput protein arrays allow the miniaturized and parallel analysis of large numbers of diagnostic markers in complex samples. Using automated colony picking and gridding, c or antibody libraries can be expressed and screened as clone arrays. Protein microarrays are constructed from recombinantly expressed, purified, and yet functional proteins, entailing a range of optimized expression systems. Antibody microarrays are becoming a robust format for expression profiling of whole genomes. Alternative systems, such as aptamer, PROfusion, nano- and microfluidic arrays are all at proof-ofconcept stage. Differential protein profiles have been used as molecular diagnostics for cancer and autoimmune diseases and might ultimately be applied to screening of high-risk and general populations. Published online: 8 May 2002 Protein arrays appear to be promising tools for drug screening and diagnostics [1]. The concept of the arrayed library allows gene expression analysis and protein interaction screening on a whole-genome scale [2]. In contrast to arrays, gene action can be studied directly, providing the proteins natural shape and functionality are maintained. This requires novel systems for high-throughput protein expression, which produce sufficient amounts of properly modified and folded molecules. Large numbers of proteins must be arrayed at high density, keeping them intact and biologically active. This is most easily achieved if molecules of the same general structure (e.g. antibodies) are arrayed. Antibody arrays are now becoming an important screening tool for a wide range of molecules in complex mixtures (e.g. body fluids). Alternatively, nucleotide aptamers might be able to mimic certain protein functions and make even more robust array formats in the future. Here we discuss the main technological challenges, recent progress and diagnostic prospects of high-throughput protein arrays (Table 1), a field that is still largely experimental but rapidly progressing. Protein features and expression systems Proteins constitute a large variety of shapes and features that govern their functions. Three-dimensional protein structures are determined by amino-acid sequences and environmental conditions. Post-translational modifications influence binding and function of many eukaryotic proteins. Glycosylation affects solubility, lifetime and activity of some proteins, such as antibodies ability to bind complement [3]. Phosphorylation regulates enzyme activities and allows specific protein protein interactions via domains recognizing phosphate groups (e.g. SH-2 domains that interact with phosphotyrosine groups in signal transduction [4]). Such post-translational modifications depend strongly on the expressed protein s cellular host. If recombinant proteins are expressed in bacteria, they are usually not glycosylated or phosphorylated, whereas S. cerevisiae tends to hyperglycosylate secreted mammalian proteins. Targeting of recombinant proteins to secretory compartments, such as the endoplasmic reticulum, allows them to form stabilizing disulfide bridges. Globular proteins consist of a hydrophilic exterior and a hydrophobic interior. If exposed to hydrophobic surfaces, organic solvents or detergents, the inside is turned out, thus rendering these proteins inactive. This emphasizes the imperative of proper handling of native proteins for functional assays and threedimensional interaction studies. Refolding denatured proteins in vitro is an arduous task, requiring customized protocols and is not attainable in many cases. Protein expression systems should be chosen carefully. E. coli is still the most convenient host organism, but many eukaryotic proteins end up in cytoplasmic /02/$ see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S (02)