Identification of complex chromosome rearrangements in the gibbon by fluorescent in situ hybridization (FISH) of a human chromosome 2q specific microlibrary, yeast artificial chromosomes, and reciprocal chromosome painting

Chromosome painting has revealed that the human chromosome homologs in lesser apes are often fragmented and translocated to a number of different hylobatid chromosomes. We investigated the fragmented human chromosome 2 homologs in gibbons to illustrate a new strategy in mapping regional and band-specific chromosomal homologies between species. Previous research showed that the DNA library specific to human chromosome 2 paints parts of four gibbon (lar species group) chromosomes (viz., 1, 10, 12, and 16) and yields five distinct hybridization signals (including two on gibbon chromosome 16). However, the exact segments of human chromosome 2 that were translocated to the various gibbon chromosomes could not be distinguished. To determine the origin of the human chromosome 2 signals, we hybridized a microlibrary for the long arm of human chromosome 2, as well as YACs specific for most of the major bands on this chromosome, to metaphases of the gibbon. For reciprocal chromosome painting, we hybridized flow-sorted gibbon chromosome probes to human chromosome 2. Each method added additional insights that helped clarify the shuffling of human chromosome 2 material in the highly reorganized gibbon genome. There was an excellent correspondence between these complementary techniques. YAC 958d2 identified the breakpoint between human chromosome 2 material present on gibbon chromosomes 10 and 16. The reciprocal chromosome painting permitted a more complete and regional assignment of homology between segments on various gibbon chromosomes to human chromosome 2. The results show that a combination of reciprocal chromosome painting, subregional microlibraries, and band-specific probes (such as YACs) can be used to identify homologies between species and to rapidly construct detailed comparative chromosome maps, especially when the karyotypes are highly rearranged.     

Human and porcine correspondence of chromosome segments using bidirectional chromosome painting

The aim of this study was to determine the correspondence between human and porcine chromosome fragments using whole chromosome painting probes from both species in heterologous hybridization experiments (bidirectional heterologous chromosome painting). Bidirectional experiments allow the determination of segment-to-segment homologies between the chromosomes of these two species. Chromosome-specific painting probes from both species were, except one, obtained by DOP-PCR or PARM-PCR amplification of flow-sorted chromosomes. The probes labeled 95% of the total length of the porcine chromosomes with human painting probes and 60% of the human chromosomes in the reverse experiments. Syntenic relationships of chromosomal segments on the karyotype of both species were determined. There was close agreement between com- parative gene mapping data and the identified homologous segments; this comparison enabled orientation of the segments. We demonstrate that bidirectional heterologous chromosome painting is a highly efficient way of generating comparative cytogenetic maps.     

Chromosome painting: a useful art

Chromosome 'painting' refers to the hybridization of fluorescently labeled chromosome-specific, composite probe pools to cytological preparations. Chromosome painting allows the visualization of individual chromosomes in metaphase or interphase cells and the identification of both numerical and structural chromosomal aberrations in human pathology with high sensitivity and specificity. In addition to human chromosome-specific probe pools, painting probes have become available for an increasing range of different species. They can be applied to cross-species comparisons as well as to the study of chromosomal rearrangements in animal models of human diseases. The simultaneous hybridization of multiple chromosome painting probes, each tagged with a specific fluorochrome or fluorochrome combination, has resulted in the differential color display of human (and mouse) chromosomes, i.e. color karyotyping. In this review, we will summarize recent developments of multicolor chromosome painting, describe applications in basic chromosome research and cytogenetic diagnostics, and discuss limitations and future directions.     

Paternally inherited chromosomal structural aberrations detected in mouse first-cleavage zygote metaphases by multicolour fluorescence in situ hybridization painting

We describe a fluorescence in situ hybridization (FISH) procedure for assessing zygotic risk of paternal exposure to endogenous or exogenous agents. The procedure employs multicolour FISH with chromosome-specific DNA painting probes plus DAPI staining for detecting both balanced and unbalanced chromosomal aberrations in mouse first-cleavage (1-Cl) zygote metaphases. Four composite probes specific for chromosomes 1, 2, 3 or X, each labelled with biotin, plus a composite probe specific for chromosome Y labelled with digoxigenin, were used. We applied this method to evaluate the effects of paternal exposure to acrylamide, a model germ cell clastogen. First-cleavage zygote metaphases, collected from untreated females mated to males whose sperm or late spermatids were treated with acrylamide, were scored for the induction of structural aberrations using both chromosome painting (PAINT analysis) and DAPI analysis. Structural chromosomal aberrations were observed in the sperm-derived, but not in the egg-derived, pronuclei. While 59.4% of the zygotes had structural aberrations by DAPI analysis, 94.1% of the same zygotes had structural aberrations by PAINT analysis (P < 0.001), illustrating the increased sensitivity for detecting translocations and insertions obtained by adding chromosome painting. These findings show that FISH painting of mouse 1-Cl zygotes when used in conjunction with DAPI analysis is a powerful model for investigating the cytogenetic defects transmitted from father to offspring.     

A case with dicentric translocation between chromosome 9 and 18: confirmation by fluorescent in situ hybridization on metaphase spread

A female child with dicentric translocation between chromosome 9 and chromosome 18 presented non-specific minor anomalies with laryngomalacia. Chromosomal analyses were performed by the G-banding method and a fluorescence in situ hybridization (FISH) technique with a specific probe for the centromeric region of chromosome 18 and the painting probe for the chromosomes 9 and 18. Her full karyotype was confirmed as 45,XX,tdic(9;18)(p24;p11). This is the first case of dicentric translocation between chromosomes 9 and 18. The FISH technique is an important tool in chromosome diagnostics.     

Identification of the chromosome 14 origin of a C-negative marker associated with a 14q32 deletion by chromosome painting

A constitutional chromosome 14 rearrangement was observed in a female with a psychodevelopmental disorder. Karyotype analysis using a variety of chromosome techniques, QFQ, GTG, CBG, Ag-NOR and DA-DAPI, showed a deletion of chromosome 14q32.1-qter region in association with a supernumerary marker chromosome. The marker, resembling a submetacentric, approximately half the size of a G group chromosome is C band and Ag-NOR negative. The heteromorphism of the satellites showed that the deleted chromosome 14 is paternal in origin. Chromosome painting using an Alu-PCR probe specific for the human chromosome 14 and fluorescent in situ hybridization (FISH) showed that the marker contains chromosome 14q32 sequences. It is likely that the marker was generated from the deleted chromosome 14 region through a complex rearrangement.     

Children with anogenital symptoms and signs referred for sexual abuse evaluations

Germ cell tumors (GCTs) are the most frequent cancer in men aged 15 to 34 years. These tumors are highly responsive to therapy with platinum-containing regimens, and 80% of cases so treated can be considered cured. Cytogenetically, 80% of GCTs have an i(12p) regardless of tumor site or histopathology, and those that are i(12p) negative have other manifestations of 12p amplification. GCTs occasionally arise extragonadally, and such cases can be especially difficult to distinguish from poorly differentiated somatic carcinomas, a situation that poses a diagnostic and treatment dilemma We developed a technique for two-color fluorescence in situ hybridization chromosome painting on nuclei released from paraffin-embedded sections. In four tumors for which GCT was a differential diagnosis, we examined the 12p and 12q chromosome arm distributions by this technique. By use of 12p and 12q painting probes developed by microdissection, 12p and 12q were distinguished and their relative distributions evaluated. In each of the four cases, 12p regions seemed to be rearranged and over-represented relative to 12q regions. In three of the cases, an apparent i(12p) could be identified. These results support a diagnosis of GCT or GCT origin in these four cases. In tumors for which specific cytogenetic abnormalities are known, chromosome painting by fluorescence in situ hybridization using paraffin-embedded tissue is a useful technique to aid in the diagnosis of tumors that are difficult to differentiate. The patients can then be placed on treatment regimens appropriate for their specific tumor type.     

Amplification of the genes BCHE and SLC2A2 in 40% of squamous cell carcinoma of the lung

Gene amplification is a common genetic change in human cancer cells. Previously, we provided the first evidence for gene amplification at chromosome band 3q26 in squamous cell lung carcinoma. In this study, the following analyses were performed: (a) we evaluated biopsies and paraffin-embedded tissues of 16 additional squamous cell lung carcinomas for gene amplification using reverse chromosome painting. Of the 16 tumors, 3 tumors showed an amplification of the entire long arm of chromosome 3, and 3 tumors showed various amplifications on 3q, all of which involved chromosome band 3q26; (b) we tested eight genes encompassing region 3q25-qter in two different tumors to identify amplified genes on chromosome 3q. The genes SI, BCHE, and SLC2A2 were amplified in both tumors; and (c) we analyzed 15 additional paraffin-embedded tissues to determine the amplification frequency of these genes. Of the 15 squamous cell lung carcinomas, 6 showed amplification for at least 1 of the genes, with BCHE and SLC2A2 as the genes most frequently amplified. Together, our reverse chromosome painting data and our PCR analysis indicate gene amplification at 3q26 in 40% of all squamous cell lung carcinomas with BCHE and SLC2A2 as possible target genes of the amplification unit in squamous cell lung carcinoma.     

Differential elimination of 3p and retention of 3q segments in human/mouse microcell hybrids during tumor growth

We have previously found that human chromosome 3 was fragmented in the course of in vivo tumor growth of monochromosomal human/mouse (A9 fibrosarcoma parent) microcell hybrids in SCID mice. Marker analysis of tumor cell lines has identified a regularly eliminated 7 cM segment on 3p21.3 referred to as the common eliminated region (CER). The same region is frequently affected by LOH in a variety of human carcinomas. The present study is a comparative chromosome painting, reverse painting, and PCR marker analysis of microcell hybrids (MCHs) that originally contained an intact chromosome 3 from two alternative donors, during and after four passages in SCID mice. We found regular elimination of 3p in parallel with preferential retention of 3q. In addition to CER on 3p, we can now define a common retained region (CRR) on 3q. It includes eight markers between D3S1282 (3q25-q26) and D3S1265 (3q27-qter) and spans approximately 43 cM. These observations are concordant with the frequent loss of corresponding 3p regions and the frequent retention, with occasional amplification, of 3q in several types of human tumors.     

A de nevo complex t(7;13;8) translocation with a deletion in the TRPS gene region

Molecular cytogenetic analyses have resolved the pathogenetic aberration of an 8-year-old girl with tricho-rhino-phalangeal syndrome type I (TRPS I), normal intelligence, and a karyotype originally described as 46,XX,t(8;13)(q24;q21). R- and Q-banding and high resolution R-banding analyses have also disclosed a seemingly mosaic abnormality of the distal short arm of chromosome 7 but have not fully characterized this abnormality. Combined primed in situ labelling and chromosome painting, and three-colour chromosome painting have revealed a complex, apparently balanced translocation t(7;13;8). Fluorescence in situ hybridization with yeast artificial chromosome and cosmid clones from 8q24.1 has shown an interstitial deletion of at least 3 Mb covering most of the TRPS I critical region.     

Ciliary beat frequency, olfaction and endoscopic sinus surgery

OBJECTIVE: This article reports that competitive hybridization using entire chromosome specific libraries as probe and human genomic DNA as the competitor allows intense and specific fluorescent staining of human chromosome in metaphase. This general approach is called "chromosome painting". METHODS: The probes comprising chromosomes 2, 5, 6, 7, 13, 14, X specific libraries were used to analyse five cases which had been suspected of subtle translocation and deletion in karyotype analysis by G-banding of metaphase cells. The authors selected entire chromosome-specific DNA libraries hybridizing with the five cases. Unlabeled human genomic DNA was used to inhibit the hybridization of sequences in the library that bind to multiple chromosome. RESULTS: The target chromosome was made at least 20 times brighter parunit length than the others. Translocations and deletions were detected clearly in metaphase and were consistent with G-banding. However, the result was clearer and the detection easier, compared with G-banding. CONCLUSION: Chromosome painting is very powerful for identification of chromosome structural aberrations. Translocation and deletion involving these chromosomes can be strikingly visualized. The hybridization intensity and specificity are such that even very small portions of the involved chromosome can be detected. This technique is especially useful in settings where high-quality banding is difficult.     

Chromosome painting in mammals as an approach to comparative genomics

Chromosome painting has become a routine tool in comparative cytogenetics. The utility of interspecies chromosome painting has been demonstrated in taxa characterized by highly rearranged karyotypes such as in rodents and lesser apes. Chromosome painting also provides a new level of precision in comparative genome analysis for eliminating errors of confounding convergence with homology. Recent results hold promise that molecular cytogenetics will make a significant contribution to the understanding of the major features of genome evolution.     

Possibilities for false-negative findings in trisomy 21 screening with FISH

In approximately 5% of individuals with Down syndrome aneuploidy results from a chromosomal rearrangement. FISH analysis on chromosome metaphases and interphase nuclei of 5 individuals with Down syndrome carrying different types of chromosome 21 translocations demonstrated the diagnostic efficiency of this method. By use of different commercially available chromosome 21 specific probes we were able to show that only the cosmid probe specific for the Down syndrome critical region (DCR) in 22qll gave reliable results for interphase analysis of trisomy 21, while the use of chromosome 21 centromere- or of painting probes carry a high risk of a false-negative diagnosis in translocation trisomy 21.     

Physics and technology of cardiovascular MR imaging

PURPOSE: Analysis of chromosomal aberrations by fluorescence in situ hybridization using a combination of chromosome painting and telomere detection in order to get more insight into: (a) the extent of incompleteness of exchanges and (b) the frequencies of interstitial fragments. MATERIALS AND METHODS: Isolated mouse splenocytes were exposed in vitro to X-rays at a dose of 2 Gy. Aberrations involving chromosomes 2 and 3 were analysed by FISH using simultaneous chromosome painting and telomere detection. RESULTS: At 2 Gy, about 10% of apparently simple exchanges are incomplete. A striking observation was the high induction of interstitial fragments, with frequencies nearly as high as that of dicentrics. Assuming, that both ends of all interstitial fragments have rejoined with each other (to form acentric rings), it can be estimated that over 92% of reactive ends of detectable breakpoints have rejoined illegitimately. Overall, equal frequencies of translocation types t(Ab) and t(Ba) (according to the PAINT nomenclature) were observed. Also, the ratios between reciprocal forms of translocations and dicentrics were close to 1 for both the chromosomes studied. CONCLUSIONS: These studies have shown that many of the frequently observed 'one-way' exchanges using painting probes, are in fact reciprocal exchanges with one participating lesion so close to the telomere that no distal signal can be detected. Frequencies of true incomplete exchanges were found to be low. Intrachanges, here detected as interstitial fragments, were observed frequently.     

Differential destabilization of repetitive sequence hybrids in fluorescence in situ hybridization

A method for painting a chromosome or chromosome region by fluorescence in situ hybridization (FISH) without blocking DNA is described. Both unique sequence and repetitive sequence components of a fluorescently labeled probe are hybridized under low-stringency conditions, but the chromosomes are washed in such a manner that repetitive sequences are differentially removed, while region-specific unique sequence fragments remain bound to the target chromosomes. We refer to this differential retention and removal of probe components as differential stability FISH.     

The adenovirus DNA binding protein enhances intermolecular DNA renaturation but inhibits intramolecular DNA renaturation

The Adenovirus DNA binding protein (DBP) imposes a regular, rigid and extended conformation on single stranded DNA (ssDNA) and removes secondary structure. Here we show that DBP promotes renaturation of complementary single DNA strands. Enhancement of intermolecular renaturation is sequence independent, can be observed over a broad range of ionic conditions and occurs only when the DNA strands are completely covered with DBP. When one strand of DNA is covered with DBP and its complementary strand with T4 gene 32 protein, renaturation is still enhanced compared to protein-free DNA, indicating that the structures of both protein-DNA complexes are compatible for renaturation. In contrast to promoting intermolecular renaturation, DBP strongly inhibits intramolecular renaturation required for the formation of a panhandle from an ssDNA molecule with an inverted terminal repeat. We explain this by the rigidity of an ssDNA-DBP complex. These results will be discussed in view of the crystal structure of DBP that has recently been determined.     

Advances in fluorescence in situ hybridization

The techniques of in situ hybridization (ISH) are widely applied for analyzing the genetic make-up and RNA expression patterns of individual cells. This review focusses on a number of advances made over the last 5 years in the fluorescence ISH (FISH) field, i.e., Fiber-FISH, Multi-colour chromosome painting, Comparative Genomic Hybridization, Tyramide Signal Amplification and FISH with Polypeptide Nucleic Acid and Padlock probes.     

Hearing aid effect in older females

We report the use of dual-colour chromosome painting to determine the exact nature of certain chromosome rearrangements observed in the pig (Sus scrofa domestica). The chromosomal abnormalities were detected by GTG- and RBG-banding techniques. The initially proposed interpretations were: (1) rcp(6;13)(p1.5;q4.1); (2) rcp(11;16)(p1.4;q1.4); (3) rcp(6;16)(p1.1;q1.1); (4) rcp(13;17)(q4.1;q1.1); (5) rcp(6;14)(q2.7;q2.1); (6) rcp(3;5)(p1.3;q2.3); (7) rcp(2; 14)(q1.3;q2.7); (8) rcp(15;17)(q1.3;q2.1). Hybridizations were carried out with biotin- and digoxigenin-labelled probes obtained by priming authorizing random mismatches polymerase chain reaction (PARM-PCR) amplification of porcine flow-sorted chromosomes. In some cases, i.e. (1), (4), (5), (6), (7) and (8), the fluorescence in situ hybridization (FISH) results allowed confirmation of the interpretations proposed with classical cytogenetic methods. Chromosome painting proved the reciprocity of the translocation in cases (1), (6) and (8), whereas modifications of the formula were proposed for case (2). Primed in situ DNA labelling (PRINS) experiments have also been carried out in case (3) using a primer specific for the centromeres of acrocentric chromosomes (first experiment) or a primer specific for the centromeres of a subset of meta- and submetacentric chromosomes including chromosome 6 (second experiment). It allowed us to demonstrate that the breakpoints occurred in the centromeric region of chromosome 16 and in the p. arm of chromosome 6, just above the centromere.     

A stable marker chromosome with a cryptic centromere: evidence for centromeric sequences associated with an inverted duplication

Centromere activation, an important mechanism in karyotype evolution, is occasionally observed in some human chromosome rearrangements. We report a possible occurrence of centromere activation in a marker chromosome containing an atypical centromere associated with an inverted duplication of the region 14q32 --> qter. The marker chromosome's reduced centromere lacks both the alpha and beta satellite sequences usually found at normal centromeres. In an attempt to identify the centromeric sequences, the marker chromosome was flow-sorted and amplified by a degenerate oligonucleotide primer polymerase chain reaction. Reverse chromosome painting experiments showed that the marker chromosome contains sequences that are unique to the distal region of chromosome 14, as well as a low copy number of (centromeric) sequences that are also highly represented in the centromeres of chromosomes 18 and 19. These data suggest the activation of a novel centromere in the 14q32 --> qter region, very likely consequent to the duplication of the region itself.     

Chromosomal rearrangements detected by FISH and G-banding

Fluorescence in situ hybridization (FISH) using chromosome-specific DNA libraries as painting probes, locus-specific unique sequence (cosmid) probes, and Y-specific repetitive sequences was applied in the analysis of eighteen cases of chromosomal rearrangements of undetermined nature. FISH clarified the origin of the extra or translocated chromosome segments in seventeen patients, one with 2q+, two with 4q+, one each with 6p+, 7p+, 9q+, 10p+, 11q+ and 12p+, two with 13q+, and one each with 15q+, 17p+, 18p+, 20p+, 21p+ and Yq+, as well as the nature of a de novo supernumerary chromosome marker in a previously reported case. By G-banding and molecular cytogenetic studies of the family members, six cases were determined to have unbalanced translocations inherited from the carrier parent. The extra translocated genetic material may cause specific trisomic syndromes, including partial 6p21.3-p23, 9q32-q34.3, 13q32-q34, 15q24-q26, and 17p11.2-p13 trisomies in those patients. A translocated 21q segment on 12p was shown by a painting probe in a patient with Down features. A patient with cat cry syndrome resulting from a loss of the terminal segment of the short arm of chromosome 5 was confirmed by a cosmid probe showing de novo reciprocal translocation between chromosomes 5 and 18:t(5;18) (p13.3;p11.31). With FISH, the extra material on the rearranged chromosome could also be identified as duplicated or translocated. The FISH technique thus provides a method for the analysis of extra structurally abnormal chromosomes (especially in de novo cases), recognizable syndromes (contiguous gene syndromes) caused by translocated deletion from parental balanced chromosome rearrangements, and supernumerary marker chromosomes. FISH subsequent to G-banding is also of great help in the confirmation of preliminary abnormal G-banded karyotypes after a modified destaining procedure. In conclusion, the combination of G-banding and FISH is very useful in the accurate diagnosis of chromosomal rearrangements.