Susanne Michel

Demystifying chromosome preparation and the implications for the concept of chromosome condensation during mitosis

The processes taking place during routine chromosome preparation are not well understood. In this study, the morphological changes in amniotic fluid cells, blood lymphocytes, and bone marrow cells in the metaphase stage were examined under an inverted microscope during chromosome preparation. The putative processes that occur during chromosome preparation were simulated in suspension, and the cells were treated with different mixtures of hypotonic solution, fixative, methanol, acetic acid, and water. Evaporation of the fixative was performed under normal atmospheric conditions and under vacuum at different levels of humidity. Freeze fracture electron microscopy was used to analyze the effects of fixative on the cell membrane. Confocal microscopic analysis was used to investigate three-dimensionally the effects of hypotonic treatment on the positions of chromosomes in fixed mitotic lymphocytes. Chromosome preparation-induced changes in the lengths of single chromosomes were also investigated. The results show that chromosome spreading involves significant water-induced swelling of mitotic cells during evaporation of the fixative from the slide, which is a prerequisite for chromosomal elongation, the production of metaphase spreads for chromosome analysis, and the appearance of Giemsa banding patterns. Hypotonic treatment is essential for well-spread metaphase chromosomes because it moves the chromosomes from a central to a more peripheral position in the cell, where they can be stretched more effectively during mitotic swelling. Like mitotic cells, isolated single chromosomes also have their own potential to swell and lengthen during chromosome preparation. We hypothesize that chromosome preparation leads to a genome-wide chromosomal region–specific opening of chromatin structures as GTG-light bands and sub-bands. Living cells may possess a similar mechanism, which is used only to open single chromatin structures to facilitate transcription. We propose the concept of chromosomal region–specific protein swelling.

The DNA-Based Structure of Human Chromosome 5 in Interphase

In contrast to those of metaphase chromosomes, the shape, length, and architecture of human interphase chromosomes are not well understood. This is mainly due to technical problems in the visualization of interphase chromosomes in total and of their substructures. We analyzed the structure of chromosomes in interphase nuclei through use of high-resolution multicolor banding (MCB), which paints the total shape of chromosomes and creates a DNA-mediated, chromosome-region-specific, pseudocolored banding pattern at high resolution. A microdissection-derived human chromosome 5-specific MCB probe mixture was hybridized to human lymphocyte interphase nuclei harvested for routine chromosome analysis, as well as to interphase nuclei from HeLa cells arrested at different phases of the cell cycle. The length of the axis of interphase chromosome 5 was determined, and the shape and MCB pattern were compared with those of metaphase chromosomes. We show that, in lymphocytes, the length of the axis of interphase chromosome 5 is comparable to that of a metaphase chromosome at 600-band resolution. Consequently, the concept of chromosome condensation during mitosis has to be reassessed. In addition, chromosome 5 in interphase is not as straight as metaphase chromosomes, being bent and/or folded. The shape and banding pattern of interphase chromosome 5 of lymphocytes and HeLa cells are similar to those of the corresponding metaphase chromosomes at all stages of the cell cycle. The MCB pattern also allows the detection and characterization of chromosome aberrations. This may be of fundamental importance in establishing chromosome analyses in nondividing cells.

Determination of the origin of single nucleated cells in maternal circulation by means of random PCR and a set of length polymorphisms

Abstract Non-invasive prenatal diagnosis on fetal nucleated erythrocytes from the maternal circulation is hampered by the small number of nucleated erythrocytes and the uncertainty as to whether they are of fetal or maternal origin. To overcome the latter limitation, single nucleated erythrocytes were separated and enriched from maternal blood by a triple density gradient and a monoclonal antibody (CD71) in combination with a magnetic activated cell sorter. Single nucleated cells were microscopically examined, individually collected with extended Pasteur pipettes, and each transferred into separate caps for the polymerase chain reaction (PCR). The DNA of the single nucleated erythrocytes was amplified at least 50-fold with a random PCR technique, viz., primer extension preamplification. Precise differentiation between maternal and fetal nucleated erythrocytes was achieved via PCR by using primers flanking highly polymorphic nucleotide repeats (D1S53, ACTBP2 and D21S11) and with a XY-specific primer pair (amelogenin). A total of 134 putative nucleated erythrocytes were analyzed from blood samples of 19 pregnant women. With the help of the polymorphic repeats, 25% were assigned as being of maternal origin, 26% of fetal origin, and 48% were uninformative. In cases with male fetuses, the amelogenin primers revealed 30% of cells to be fetal nucleated erythrocytes, the remaining 70% being of maternal origin. The results indicate that the combination of random PCR and PCR-mediated polymorphism analysis on the DNA of single nucleated erythrocytes is a useful technique for non-invasive prenatal diagnosis.

Proteome analysis of maternal serum samples for trisomy 21 pregnancies using ProteinChip arrays and bioinformatics

A surface-enhanced laser desorption/ionization time of flight (SELDI-TOF)-based ProteinChip System was used as a tool for rapid discovery and identification of protein patterns in serum that discriminate between trisomy 21 and unaffected pregnancies. We analyzed 24 serum samples from women carrying a trisomy 21 pregnancy and 32 with an unaffected pregnancy, ranging from 10.0 to 14.0 weeks of gestation. The resulting protein profiles were submitted to a clustering algorithm, a rule extraction, a rating, and a rule base construction step. For the generated combined rule base, the specificity and sensitivity for the prediction of a trisomy 21 pregnancy reach 97% and 91%, respectively.

Characteristic genomic imbalances in pediatric pheochromocytoma

Pheochromocytoma (PCC) in children is rare, genetically not well described, and often related to a poor prognosis. We detected genomic imbalances in all 14 tumors from children analyzed by comparative genomic hybridization. A combinatorial loss of chromatin from 3p and 11p was a common feature in 10 of 14 (72%) patients, which was a result of either a loss of a total chromosome 3 and a total chromosome 11 in 6 of 10 patients, or confined deletions of their p arms in 4 of 10 patients. All patients exhibiting a loss of 3p and 11p carried VHL mutations. The VHL mutations were constitutive in 9 cases and somatic and restricted to tumor DNA in the remaining tumor. On the other hand, VHL mutations were absent in 4 patients, 2 who had other familial syndromes (NF1, SDHD) and 2 with unknown etiology. Our data show that the pattern of imbalances in the tumor DNA of PCC patients strongly correlated with an underlying familial VHL mutation. Furthermore, we show that true sporadic PCC is rare in childhood. Thus, children with PCC should be checked for a related predisposing gene. This would also identify familial syndrome patients requiring long‐term monitoring for other syndrome‐related malignancies.

Multicolor FISH used for the characterization of small supernumerary marker chromosomes (sSMC) in commercially available immortalized cell lines.

There are only about 30 commercially available cell lines which include small supernumerary marker chromosomes (sSMC). As approximately 2.5 million people worldwide are carriers of an sSMC, this small number of immortalized cell lines is hard to understand. sSMC cell lines provide practically unlimited material for continuing studies e.g. to learn more about marker chromosome formation, or karyotypic evolution. To obtain information about their genetic content, in the present study we analyzed by FISH and multicolor-FISH approaches 19 sSMC cell lines obtained from the European Collection of Cell Cultures (ECACC). Microdissection and reverse painting, (sub-) centromere-specific multicolor-FISH (sub-)cenM-FISH, multicolor banding (MCB) and selected locus-specific FISH probes were applied. Thus, we were able to characterize comprehensively 14 out of 19 sSMC carrying cell lines; in the remaining five cases an sSMC could not be detected. Surprisingly, in six of the nine cell lines with sSMC previously characterized for their chromosomal origin by others, those results had to be revised. This has impact on the conclusions of previous studies, e.g. for uniparental disomy (UPD) in connection with sSMC.

Precise breakpoint characterization of the colon adenocarcinoma cell line HT-29 clone 19A by means of 24-color fluorescence in situ hybridization and multicolor banding.

In a previous issue of this journal, we read with interest the article of Kawai et al. (2002) regarding the karyotype analysis of the colon adenocarcinoma cell line HT-29. To study this karyotype, the authors combined G-banding, fluorescence in situ hybridization (FISH), and spectral karyotyping (SKY). As mentioned by Kawai et al. (2002), Gbanding analysis for HT-29 had been carried out before by Chen et al. (1987) and Bertrand et al. (1999). In addition, however, there are two other previous attempts to determine the karyotype of HT-29, through use of a combination of comparative genomic hybridization (CGH) and SKY (Ghadimi et al., 2000; Abdel-Rahman et al., 2001). As demonstrated recently, the multicolor banding (MCB; according to Liehr et al., 2002a) method allows the characterization of exactly the breakpoints and the orientation of chromosomal rearrangements (Weise et al., 2002), even in highly rearranged karyotypes (Mrasek et al., 2001). Thus, a detailed analysis of aberrations and characterization of breakpoints in HT-29 clone 19A, which shares the main chromosomal aberrations with HT-29, was performed by MCB. HT-29 clone 19A (terminally differentiated and described by Augeron and Laboisse, 1984) differs according to our data presented below from the HT-29 cell line described by Kawai et al. (2002) in 10 chromosomal aberrations. Furthermore, the HT-29 clone described by Abdel-Rahman et al. (2001) differs from the one investigated by Kawai et al. (2002) by 10 rearrangements. After an orienting classification of chromosomal rearrangements by 24-color FISH as described by Kuechler et al. (2002), MCB was performed on all chromosomes involved in rearrangements in HT-29 clone 19A (Fig. 1). When necessary for exact characterization of rearrangements and/or origin of derivative chromosomes, MCB analysis was complemented by centromere-specific probes (chromosomes 1, 2, 4, 6, 7, 8, 9, 12, 13, 17, 18, 19, and 22), locus-specific probes (MYC and TP53), partial (chromosome arms 3p, 3q, 12p, 17q, and 18p), and whole chromosome painting probes (chromosomes 2, 3, 4, 5, 6, 11, 16, 17, 18, 19, and 22). At least 10 metaphases were evaluated per hybridized probe or probe set. On the basis of the combination of 24-color FISH, MCB, and the additional FISH probes, a composite karyotype could be described (Table 1). A survey of hybridization results of all aberrant and corresponding normal chromosomes is given in Figure 1. The composite karyotype after MCB contained 20 different aberrant chromosomes, with a total number of 36 breakpoints, 33 of which could be characterized in detail. The der(22)t(17;22;17), with high probability identical to marker M9 described by Kawai et al. (2002), was the only marker that could not be fully characterized. In concordance with the literature, all detected structural aberrations were unbalanced and almost all human chromosomes were involved, apart from chromosomes 10, 14, 15, and 21. However, Kawai et al. (2002) and Ghadimi et al. (2000) reported a t(6;14) and a der(6)t(6;14), respectively. The latter reflects the heterogeneity of this very common cell line. Our HT-29 clone 19A and the HT-29 cell line studied by Kawai et al. (2002) presented with a similar copy number for most of the chromosomes. Differences were that HT-29 contained the marker chromosomes M1, M10, M11, M13, M15, M17, and M18, which were not present in clone 19A. However, our clone showed an additional marker similar to M12 (Chen et al., 1987), a der(5)t(5;19), and a der(11)t(11; 20). A der(2)t(1;2), also described in HT-29 by AbdelRahman et al. (2001), a der(3)ins(3;12), also detected in HT-29 by Ghadimi et al. (2000), and a der(4)t(2;4) were present instead of a normal chromosome 2, M1, or M17, respectively. Applying MCB, breakpoints could be confirmed for M2, M4, and M14 and further characterized for M6 and M16. Additionally, the huge derivative (8q)-isochromosome (M6) was demonstrated by MCB to include a homogeneously staining region (HSR) derived from 8q24, which was subsequently verified to consist of a MYC amplicon (Fig. 1).

DNA degradation during maturation of erythrocytes – molecular cytogenetic characterization of Howell-Jolly bodies

Howell-Jolly bodies (HJBs) are small DNA-containing inclusions of erythrocytes and are often present after splenectomy. The genetic composition of HJBs is unknown at present. We isolated individual erythrocytes that had inclusion bodies from five splenectomized patients and performed DNA amplification using degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) with subsequent reverse painting on normal male metaphase spreads. We also measured the sizes of HJBs in erythrocytes from a splenectomized patient using an inverted microscope. Two-dimensional positions of HJBs were projected onto a virtual erythrocyte. The average size of HJBs was 0.73 ± 0.17 µm (range 0.4–1.1 µm). Inside the erythrocyte the HJBs were found to be equally distributed. Small HJBs contained DNA from one or two centromeres and larger HJBs contained DNA from up to eight different centromeres. Centromeric DNA from chromosomes 1/5, 7, 8, and 18 was most frequently observed. Signals from the centromeric regions of chromosomes 3, 4, 9, and 10 were not observed. Signals from euchromatic regions were detected in a few cases. We hypothesize that in addition to enucleation and nucleus fragmentation DNA degradation during maturation of erythrocytes preferentially eliminates euchromatic DNA. Similarities between these processes and those described for embryonic stem cells suggest that most stem cells are able to degrade DNA in a genetically controlled manner.

Microdissection-derived Murine Mcb Probes from Somatic Cell Hybrids

The multicolor-banding (mcb) technique is a fluorescence in situ hybridization (FISH)-banding approach, which is based on region-specific microdissection libraries producing changing fluorescence intensity ratios along the chromosomes. The latter are used to assign different pseudocolors to specific chromosomal regions. Here we present the first three available mcb-probe sets for the Mus musculus chromosomes 3, 6, and 18. In the present work, the creation of the microdissection libraries was done for the first time on mouse/human somatic cell hybrids. During creation of the mcb-probes, the latter enabled an unambiguous identification of the, otherwise in GTG-banding, hardly distinguishable murine chromosomes.

The hierarchically organized splitting of chromosome bands into sub-bands analyzed by multicolor banding (MCB)

To clarify the nature of chromosome sub-bands in more detail, the multicolor banding (MCB) probe-set for chromosome 5 was hybridized to normal metaphase spreads of GTG band levels at ∼850, ∼550, ∼400 and ∼300. It could be observed that as the chromosomes became shorter, more of the initial 39 MCB pseudo-colors disappeared, ending with 18 MCB pseudo-colored bands at the ∼300-band level. The hierarchically organized splitting of bands into sub-bands was analyzed by comparing the disappearance or appearance of pseudo-color bands of the four different band levels. The regions to split first are telomere-near, centromere-near and in 5q23→q31, followed by 5p15, 5p14, and all GTG dark bands in 5q apart from 5q12 and 5q32 and finalized by sub-band building in 5p15.2, 5q21.2→q21.3, 5q23.1 and 5q34. The direction of band splitting towards the centromere or the telomere could be assigned to each band separately. Pseudo-colors assigned to GTG-light bands were resistant to band splitting. These observations are in concordance with the recently proposed concept of chromosome region-specific protein swelling.