Heinz-Ulli G. Weier

Multicolor chromosome banding (MCB) with YAC/BAC-based probes and region-specific microdissection DNA libraries

Multicolor chromosome banding (MCB) allows the delineation of chromosomal regions with a resolution of a few megabasepairs, i.e., slightly below the size of most visible chromosome bands. Based on the hybridization of overlapping region-specific probe libraries, chromosomal subregions are hybridized with probes that fluoresce in distinct wavelength intervals, so they can be assigned predefined pseudo-colors during the digital imaging and visualization process. The present study demonstrates how MCB patterns can be produced by region-specific microdissection derived (mcd) libraries as well as collections of yeast or bacterial artificial chromosomes (YACs and BACs, respectively). We compared the efficiency of an mcd library based approach with the hybridization of collections of locus-specific probes (LSP) for fluorescent banding of three rather differently sized human chromosomes, i.e., chromosomes 2, 13, and 22. The LSP sets were comprised of 107 probes specific for chromosome 2, 82 probes for chromosome 13, and 31 probes for chromosome 22. The results demonstrated a more homogeneous coverage of chromosomes and thus, more desirable banding patterns using the microdissection library-based MCB. This may be related to the observation that chromosomes are difficult to cover completely with YAC and/or BAC clones as single-color fluorescence in situ hybridization (FISH) experiments showed. Mcd libraries, on the other hand, provide high complexity probes that work well as region-specific paints, but do not readily allow positioning of breakpoints on genetic or physical maps as required for the positional cloning of genes. Thus, combinations of mcd libraries and locus-specific large insert DNA probes appear to be the most efficient tools for high-resolution cytogenetic analyses.

Spectral imaging in preconception/preimplantation genetic diagnosis of aneuploidy: multicolor, multichromosome screening of single cells.

Purpose:Our purpose was to evaluate the utility of spectral imaging for multicolor, multichromosome enumeration in human interphase cell nuclei.Methods:Chromosome-specific probes labeled with different fluorochromes or nonfluorescent haptens were obtained commercially or prepared in-house. Metaphase spreads, interphase lymphocytes, or blastomeres cells were hybridized with either 7 or 11 distinctly different probes. Following 46 hr of hybridization, slides were washed and detected using either a filter-based quantitative image processing system (QUIPS) developed in-house or a commercial spectral imaging system.Results:The filter-based fluorescence microscope system is preferred for simultaneous detection of up to seven chromosome targets because of its high sensitivity and speed. However, this approach may not be applicable to interphase cells when 11 or more targets need to be discriminated. Interferometer-based spectral imaging with a spectral resolution of approximately 10 nm allows labeling of chromosome-specific DNA probes with fluorochromes having greatly overlapping emission spectra. This leads to increases in the number of fluorochromes or fluorochrome combinations available to score unambiguously chromosomes in interphase nuclei.Conclusions:Spectral imaging provides a significant improvement over conventional filter-based microscope systems for enumeration of multiple chromosomes in interphase nuclei, although further technical development is necessary in its application to embryonic blastomeres. When applied to preconception/preimplantation genetic diagnosis, presently available probes for spectral imaging are expected to detect abnormalities responsible for 70–80% of spontaneous abortions caused by chromosomal trisomies.

Detection of chromosome translocation products in single interphase cell nuclei.

Publisher Summary Chromosome translocations occur at a frequency of about 0.1% in the general population. Translocation carriers experience a higher incidence of infertility and reproductive failures and also a higher risk of conceiving chromosomally abnormal offspring. Patients who are carriers of Robertsonian or reciprocal translocations may benefit from in vitro fertilization (IVF) followed by preconception and preimplantation genetic diagnosis (PGD). Three approaches for PGD of structural abnormalities based on fluorescence in situ hybridization (FISH) are discussed in this chapter: chromosome painting of polar bodies, probes mapping distal to the break points, and probes spanning the break points. When the carrier is female, the chromosomally normal versus abnormal oocytes can be determined by using whole chromosome painting probes hybridized to the first polar body chromosomes. When the carrier is male, the only method available for PGD is the blastomere biopsy after in vitro fertilization (IVF) and in vitro culture of the embryo. The chapter describes rapid and inexpensive procedures to prepare case-specific probes for FISH-based PGD in germ cells or embryos by using yeast artificial chromosomes (YACs) as probes spanning or flanking translocation break points.

Fluorescence in situ hybridization and spectral imaging analysis of human oocytes and first polar bodies.

We investigated the frequencies of abnormalities involving either chromosome 1, 16, 18, or 21 in failed-fertilized human oocytes. Although abnormalities involving chromosome 16 showed an age-dependent increase, results for the other chromosomes did not show statistically significant differences among the three age groups, <35 years, 35–39 years, and >39 years. The scoring of four chromosomes is likely to underestimate the true rate of aneuploid cells. Therefore, for a pilot study investigating a more-comprehensive analysis of oocytes and their corresponding first polar bodies, we developed a novel eight-probe chromosome enumeration scheme using fluorescence in situ hybridization and spectral imaging analysis.

Somatic Genomic Variations in Extra-Embryonic Tissues

In the mature chorion, one of the membranes that exist during pregnancy between the developing fetus and mother, human placental cells form highly specialized tissues composed of mesenchyme and floating or anchoring villi. Using fluorescence in situ hybridization, we found that human invasive cytotrophoblasts isolated from anchoring villi or the uterine wall had gained individual chromosomes; however, chromosome losses were detected infrequently. With chromosomes gained in what appeared to be a chromosome-specific manner, more than half of the invasive cytotrophoblasts in normal pregnancies were found to be hyperdiploid. Interestingly, the rates of hyperdiploid cells depended not only on gestational age, but were strongly associated with the extraembryonic compartment at the fetal-maternal interface from which they were isolated. Since hyperdiploid cells showed drastically reduced DNA replication as measured by bromodeoxyuridine incorporation, we conclude that aneuploidy is a part of the normal process of placentation potentially limiting the proliferative capabilities of invasive cytotrophoblasts. Thus, under the special circumstances of human reproduction, somatic genomic variations may exert a beneficial, anti-neoplastic effect on the organism.

Bioinformatic Tools Identify Chromosome-Specific DNA Probes and Facilitate Risk Assessment by Detecting Aneusomies in Extra-embryonic Tissues.

Despite their non-diseased nature, healthy human tissues may show a surprisingly large fraction of aneusomic or aneuploid cells. We have shown previously that hybridization of three to six non-isotopically labeled, chromosome-specific DNA probes reveals different proportions of aneuploid cells in individual compartments of the human placenta and the uterine wall. Using fluorescence in situ hybridization, we found that human invasive cytotrophoblasts isolated from anchoring villi or the uterine wall had gained individual chromosomes. Chromosome losses in placental or uterine tissues, on the other hand, were detected infrequently. A more thorough numerical analysis of all possible aneusomies occurring in these tissues and the investigation of their spatial as well as temporal distribution would further our understanding of the underlying biology, but it is hampered by the high cost of and limited access to DNA probes. Furthermore, multiplexing assays are difficult to set up with commercially available probes due to limited choices of probe labels. Many laboratories therefore attempt to develop their own DNA probe sets, often duplicating cloning and screening efforts underway elsewhere. In this review, we discuss the conventional approaches to the preparation of chromosome-specific DNA probes followed by a description of our approach using state-of-the-art bioinformatics and molecular biology tools for probe identification and manufacture. Novel probes that target gonosomes as well as two autosomes are presented as examples of rapid and inexpensive preparation of highly specific DNA probes for applications in placenta research and perinatal diagnostics.

Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

BackgroundFluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100 kb, careful probe selection and characterization are of paramount importance.ResultsWe describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BACs can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific ~6 kb plasmid onto an unusually small, ~55 kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-κB2 locus.ConclusionThe relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

Quantitative DNA fiber mapping.

The assembly of sequence ready, high-resolution physical maps and construction of minimally overlapping contigs for the human as well as model genomes requires accurate determination of the extent of overlap between adjacent clones as well as their relative orientation. This is presently done by procedures such as clone fingerprinting, Southern blot analysis or clone end sequencing. We present a complementary analytical technique to map directly cloned DNA sequences on to individual stretched DNA molecules. This approach uses the hydrodynamic force of a receding meniscus to prepare straight high molecular weight DNA molecules that provide a linear template of approximately 2.3 kb/microns on to which the cloned probes can be mapped by in situ hybridization. This technique has numerous advantages such as a very high density of mapping templates, reproducible stretching of the mapping template providing a linear genomic scale, determination of clone orientation and direct visualization of DNA repeats. The utility and accuracy of quantitative DNA fiber mapping are illustrated through three examples: (i) mapping of lambda DNA restriction fragments along linearized approximately 49 kb long lambda phage DNA molecules with approximately 1 kb precision; (ii) localization of the overlap between a cosmid and a colinear P1 clone; and (iii) mapping of P1 clones along an approximately 490 kb yeast artificial chromosome (YAC) with approximately 5 kb precision and estimation of the approximately 25 kb gap between them.

High-performance analysis of single interphase cells with custom DNA probes spanning translocation break points

The chromatin organization of interphase cell nuclei, albeit an object of intense investigation, is only poorly understood. In the past, this has hampered the cytogenetic analysis of tissues derived from specimens where only few cells were actively proliferating or a significant number of metaphase cells could be obtained by induction of growth. Typical examples of such hard to analyze cell systems are solid tumors, germ cells and, to a certain extent, fetal cells such as amniocytes, blastomeres or cytotrophoblasts. Balanced reciprocal translocations that do not disrupt essential genes and thus do not led to disease symptoms exit in less than one percent of the general population. Since the presence of translocations interferes with homologue pairing in meiosis, many of these individuals experience problems in their reproduction, such as reduced fertility, infertility or a history of spontaneous abortions. The majority of translocation carriers enrolled in our in vitro fertilization (IVF) programs carry simple translocations involving only two autosomes. While most translocations are relatively easy to spot in metaphase cells, the majority of cells biopsied from embryos produced by IVF are in interphase and thus unsuitable for analysis by chromosome banding or FISH-painting. We therefore set out to analyze single interphase cells for presence or absence of specific translocations. Our assay, based on fluorescence in situ hybridization (FISH) of breakpoint-spanning DNA probes, detects translocations in interphase by visual microscopic inspection of hybridization domains. Probes are prepared so that they span a breakpoint and cover several hundred kb of DNA adjacent to the breakpoint. On normal chromosomes, such probes label a contiguous stretch of DNA and produce a single hybridization domain per chromosome in interphase cells. The translocation disrupts the hybridization domain and the resulting two fragments appear as physically separated hybridization domains in the nucleus. To facilitate the detection, DNA probes for breakpoints on different chromosomes are labeled in different colors, so the translocation event can be detected as a fusion of red and green hybridization domains. We applied this scheme successfully for the analysis of somatic and germ cells from more than 20 translocation patients, each with individual breakpoints, and provide summaries of our experience as well as strategies, cost and time frames to prepare case-specific translocation probes.

Simultaneous scoring of 10 chromosomes (9,13,14,15,16,18,21,22,X, and Y) in interphase nuclei by using spectral imaging

Numerical aberrations involving parts of or entire chromosomes have detrimental effects on mammalian embryonic, and perinatal development. Only few fetuses with chromosomal imbalances survive to term, and their abnormalities lead to stillbirth or cause severely altered phenotypes in the offspring (such as trisomies involving chromosomes 13, 18, 21, and anomalies of X, and Y). Because aneuploidy of any of the 24 chromosomes will have significant consequences, an optimized preimplantation and prenatal genetic diagnosis (PGD) test will score all the chromosomes. Since most cells to be analyzed will be in interphase rather than metaphase, we developed a rapid procedure for the analysis of interphase cells such as lymphocytes, amniocytes, or early embryonic cells (blastomeres). Our approach was based on in situ hybridization of chromosome-specific non-isotopically labeled DNA probes and Spectral Imaging. The Spectral Imaging system uses an interferometer instead of standard emission filters in a fluorescence microscope to record high resolution spectra from fluorescently stained specimens. This bio-imaging system combines the techniques of fluorescence optical microscopy, charged coupled device imaging, Fourier spectroscopy, light microscopy, and powerful analysis software. The probe set used here allowed simultaneous detection of 10 chromosomes (9, 13, 14, 15, 16, 18, 21, 22, X, Y) in interphase nuclei. Probes were obtained commercially or prepared in-house. Following 16 - 40 h hybridization to interphase cells and removal of unbound probes, image spectra (range 450 - 850 nm, resolution 10 nm) were recorded and analyzed using an SD200 Spectral Imaging system (ASI, Carlsbad, CA). Initially some amniocytes were unscoreable due to their thickness, and fixation protocols had to be modified to achieve satisfactory results. In summary, this study shows the simultaneous detection of at least 10 different chromosomes in interphase cells using a novel approach for multi-chromosome analysis.